Intro to Proteomics 1.0

Tuesday AM Lecture 1: Intro to Proteomics 1.0

Tim Griffin CMSP Faculty Director Professor, BMBB

For participants: • Learn basics of MS-based proteomics • Learn what’s necessary for success using MS-based proteomics • Designing experiments; sample preparation; data analysis

For CMSP staff: • Prepare users so they are equipped to have success working with CMSP • Manage expectations – what can these technologies do and not do

CMSP Participants

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 5 Terminology made sensible Stage-tip Ion trap b-ion MS/MS iTRAQ TOF NanoLC HCD quadrupole Right??? on! Precursor ion monoisotopic MALDI ESI

Participants

CMSP

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 6 Who we are Center for Mass Spectrometry and Proteomics

• Operated through the Department of Biochemistry, Molecular Biology and Biophysics • Serving biological MS-related research needs across UofM campus and external institutions/private companies • Fee-for-service Internal Service Organization (ISO) • Supported by all Colleges at UofM using CMSP and Office of Vice President for Research (plus variety of granting sources) • Extensive collaboration with Minnesota Supercomputing Institute/OIT/UMII

• Primary mission to support research efforts at the University of Minnesota, but also train others in the use of advanced technologies and research approaches

Julie Kirihara Manager

Informatics Analyst Candace Guerrero PostdoctoralResearch Associate researcher • 150+ collective years of experience in biological MS; hundreds of scientific publications • Diverse expertise – design, sample preparation, instrumentation, data analysis • Experience with MANY sample types and research studies • Fish….gophers….periodontal bacteria…snake venom © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 8 ‘Omic technologies and the molecular biology paradigm

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 9 Why proteomics and direct protein analysis? (Genomic sequencing is cheaper, faster, more comprehensive…why proteomics?)

• DNA/RNA characterization cannot predict post-transcriptional events

“Proteomics includes not only the identification and quantification of proteins, but also the determination of their localization, modifications, interactions, activities, and, ultimately, their function.” -Stan Fields in Science, 2001.

Alternatively: proteomics = high-throughput biochemistry

• measurement of protein response, which is not always indicated by mRNA response • post-translational modifications • macromolecular interactions • sub-cellular location • high-resolution structural and molecular characterization • integration with genomic/transcriptomic data to comprehensively characterize biological systems

• two-dimensional gel electrophoresis • mass spectrometry • protein chips • yeast 2-hybrid • phage display • antibody engineering • high-throughput protein expression • high-throughput X-ray crystallography • cell imaging

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 13 Enabling MS-based proteomics: “soft” ionization

• Making large, non-volatile biomolecules fly

Electrospray ionization Matrix-assisted laser desorption/ionization (ESI) (MALDI)

+ + - - ionization separation by m/z* detection - + + - + +  MALDI  quadrupole  mass analysis of  Electrospray:  ion trap proteins, peptides liquid chromatography  time-of-flight nanospray *m/z = mass-to-charge

Image from : http://www.medwow.com

Image from: https://www.sdstate.edu/chem/mass-spec

Image from: http://planetorbitrap.com Image from: http://planetorbitrap.com

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 16 Example of technology progress: more sensitive MS

Image from ASMS 2014 workshop (Speaker: Haas)

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 17 The information currency of MS Relative Abundance

200 400 600 800 1000 1200 m/z

m/z separation and detection

m/z separation

m/z separation and detection

ionization

Biological inquiry Hypothesis Sample Experimental design MS analysis Data analysis preparation

• Workshop structured to follow this ordering • All aspects are important: each must be done well for success • Challenge: • technologies within each component always changing • interdisciplinary

• Garbage in, garbage out

•Protein mixtures isolated from biological sources are complex (hundreds to thousands of components)

• Mass spectrometers have limited peak capacities requiring separation and fractionation of protein and peptide mixtures prior to analysis

• Separation methods include: • gels • liquid chromatography • affinity chromatography • immunochromatography • selective enrichment by covalent chemistry © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 21 Protein chemistry: a challenge

• Proteins offer unique challenges compared to other biomolecules (e.g. nucleic acids):

– Solubility

– Abundance (no PCR!)

– Chemical heterogeneity Each protein is a unique character!

• Separating molecular mixtures prior to introduction into MS

organic concentration in mobile phase base peak intensity

time

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 23 Some example applications: from simple to complex

The “simple”: identifying a gel separated protein

2D gel electrophoresis: the original proteomics technology…but how to ID proteins?

Gygi, et. al. 1999, Molecular and Cellular Biology 19:1720

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 24 Even the simple still requires care….. • Process of identifying a gel-separated protein

In-gel digestion

Peptide purification Gygi, et. al. 1999, Molecular and Cellular Biology 19:1720

LC-MS/MS

Sequence Database Searching

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 25 A bit more complicated: Identifying PTMs on a protein

• Phosphorylation • Glycosylation • Oxidations • Acetylation • Methylation • Lipid anchors • Ubiquitinylation/sumoylation ……

etc

BUT….PTM analysis is not necessarily routine or easy!!

(abundance, enrichment, ionization, fragmentation….)

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 26 Still more complicated: identification of proteins in complex mixtures • More complicated sample preparation (fractionation)

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 27 A bit more complicated: Quantitative proteomics

S. cerevisiae cell cycle (compliments of J.A. Huberman)

• Protein abundance is dynamic in response to environmental, genetic, biochemical, pathological perturbations.

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 28 Quantitative proteomics: many methods available • Labeled versus un-labeled

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 30 Dealing with the data: the rate-limiting step?

Data acquisition

Raw data processing (Database searching)

Analysis of processed data (Statistical filtering, quantitative analysis)

Data organization and interpretation

Archiving and databasing

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 32 Bioinformatic interpretation and hypothesis generation

KEGG pathways

Good luck

May all your ions fly well!

Experimental Design Outline Terminology

• Goals • Targeted vs. Discovery • Designing your • Dynamic range experiment • Inference • Experimental design • Sampling error • Workflow • Bias • Challenges • Variability • Tradeoffs • Blocking • Randomization

Proteomics Research Areas

• Expression proteomics – Protein expression and regulation, including identification, quantification, differential expression, e.g., compare physiological or environmental conditions • Functional proteomics – Protein function and protein interaction networks with other proteins, lipids, DNA, ligands • Structural proteomics – Studying and modeling protein structure e.g., inferring function from protein structure

https://doi.org/10.1093/beheco/aru096 Common Goals

• Protein identification • Protein-protein interactions via immunoprecipitation (IP) or co-IP • Post translational modifications • Protein quantification, relative or absolute Experimental Design

• Design your experiments with a particular biological question in mind • Plan an exploratory survey or a hypothesis-driven question • Plan your experimental design before you carry out an experiment Design Choices • Discovery vs. Targeted • Workflow Technology • Tradeoffs • Statistical Rigor

e.g., Design an Experiment to measure Protein-Protein Interactions

Co-IP with FLAG- NEGATIVE tagged bait protein CONTROL 1. Co-IP with fusion protein 2. Co-IP with FLAG peptide (negative control) 3. Elute protein complexes, run SDS-PAGE 4. In-gel trypsin digestion of protein bands of interest 5. Protein identification by peptide mass spectrometry 6. Repeat or use biological replicates

PMID 19170023 Possible outcomes for protein identification by peptide mass spectrometry

High Protein Sequence Coverage

Low Protein Sequence Coverage

• Consider follow-up experiments Protein Quantification: e.g., Abundance Changes over Time

Proteome dynamics of fate changes in Embryonic Stem Cells

• Mass Spectrometry data shows relative protein abundance changes over time

Ref: Raltelaar, et al., Nature Reviews Genetics 14, 35-48 (January 2013) TMT* 10-plex Experimental Design Relative Protein Quantification

REFERENCE CATERGORY INFECTED CATEGORY 5 biological replicates uninfected 5 biological replicates infected w/Bacteria

126 127N 127C 128N 128C 129N 129C 130N 130C 131

• Biological replicates reduce sampling error and increase measurement precision • Numbers of samples are balanced across conditions

* TMT = tandem mass tag (Thermo Scientific) isotope labeling kit for protein relative quantification Relative Protein Quantification with Stable Isotopes (iTRAQ 8plex) Experimental Design: 3 Sets of Biological Replicates

Experiment 1 Experiment 2 Experiment 3 Biological Biological Biological Condition Sample Condition Sample Condition Sample CONTROL B1 CONTROL B7 CONTROL B13 Drug 1 B2 Drug 1 B8 Drug 1 B14 Drug 2 B3 Drug 2 B9 Drug 2 B15 Drug 3 B4 Drug 3 B10 Drug 3 B16 Drug 4 B5 Drug 4 B11 Drug 4 B17 Placebo B6 Placebo B12 Placebo B18

Isotope Isotope Isotope Isotope Isotope Isotope Isotope Isotope 113 114 115 116 117 118 119 121 Experiment 1 POOL Ref 01 CONTROL DRUG 2 DRUG 4 Placebo DRUG 1 POOL Ref 02 DRUG 3

Experiment 2 CONTROL POOL Ref 03 DRUG 1 Placebo POOL Ref 04 DRUG 4 DRUG 3 DRUG 2

Experiment 3 DRUG 2 DRUG 1 POOL Ref 05 DRUG 3 CONTROL DRUG 4 Placebo POOL Ref 06

• Pooled reference: mix equal aliquots of biological samples B1 – B18 • Randomize the ISOTOPE LABELS across samples per experiment • BLOCK SAMPLES identically across experiments Typical Mass Spectrometry-Based Proteomics Workflow

Sample Prep

discovery Mass Spec or targeted Experimental Design Experimental Data Analysis

REF: Adapted from Walther T, Mann M. JCB 2010; 190: 491-500 The Mass Spec Inference Problem

• A molecule that is PRESENT in a sample could be UNDETECTABLE by mass spectrometry – Causes • High dynamic range of protein content • Sample preparation methods • Instrumentation choice • Data analysis methods • And more…. • Identification and quantification requires statistics, which requires experimental design Inference challenges & mass spectrometry

“Schematic representation of the fraction of a proteome that can by identified or quantified by mass-spectrometry-based proteomics. Cellular proteins span a wide range of expression and current mass spectrometric technologies typically sample only a fraction of all the proteins present in a sample. Due to limited data quality, only a fraction of all identified proteins can also be reliably quantified” PMID: 17668192 Tradeoffs Number of analytes vs. number of samples

(Discovery) (Targeted)

http://www.sepscience.com/Sectors/Proteomics/Articles/460-/Advances-in-Clinical-Proteomics--A-Workshop-Report Sources of Bias and Variability

gender individual

lifestyle age Population diet Biological

batch effects: column age technique Instrument solvent variation Sample Handling storage age ambient air difficulty personnel preventative maintenance schedule Control for Bias & Variability in Quantification Experiments • Appropriate number of biological samples • Appropriate number of technical Replicates • Normalization – corrects for systematic differences in the total amount of protein across groups in a multi-group comparison – e.g., divide each quant value by the median intensity value • Blocking • Randomization Control for Bias & Variability in Quantification Experiments • Control for Bias – Control group – Randomization – Normalization • Reduce effects of sampling error – Replication – Balance (equal number of sample groups) – Blocking (identical replication of treatment groups/categories in different settings)

Why Variance Matters Example Hypothesis: the amount of ‘Analyte 3’ is different between samples A and C Scenario 1 Scenario 2 Normalization (control for sample prep and instrument bias)

Block design (example) Randomization

Ref: Institute of Biostatistics and Analyses Masaryk University Workflow Technologies

Sample Prep Data Analysis

Mass Spec

Ref: Mallick & Kuster, Nature Biotechnology, 28, 695–709. (2010) Before you start …

PLAN

your experiments carefully

• Discuss the project with CMSP personnel • Consult a statistician when necessary Tuesday AM Lecture 3: Proteomic Sample Preparation Basics

Todd Markowski

Bottom Up Proteomics

Proteolytic HPLC

Digestion or SPE Proteins Peptides Mass Spec. Analysis

Top Down Proteomics

HPLC, Gelfree, CE

Proteins FT-ICR Analysis Mass Spec.

adpated from http://www.piercenet.com/method/sample-preparation-mass-spectrometry © 2015 Regents of the University of Minnesota. All rights reserved. 60 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Sample preparation is one of the most important aspects of any successful proteomics experiment! Questions you should ask before proteomics sample prep:

1. How should I collect the samples? Mass Consistency, consistency… Spec. ...and more consistency Sampling for biological variation Document all steps from sample collection thru prep – Good lab notes! 2. What do I hope to analyze? Simple protein ID Differentially expressed proteins (ITRAQ, SILAC, label free or targeted) Post-translational modifications (PTM) Cytoplasmic, membrane, nuclear, mitochondrial, etc. Immunoprecipitation/binding partners

3. How do I best extract my proteins of interest? In-gel: SDS-PAGE (1D or 2D) In-solution Enrichment/affinity strategies Depletion of high abundant proteins

4. Is the extraction buffer compatible with mass spectrometry? If not, how do I get rid of problematic buffer components? Solid Phase Extraction (SPE), detergent removal spin-column Cloud point extraction of non-ionic detergents Ethyl acetate extraction of detergents Protein precipitation Dialysis, molecular weight cut-off spin filter

© 2015 Regents of the University of Minnesota. All rights reserved. 62 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Overview: Sample Prep to Mass Spectrometry Work Flow

2DE

Image: http://www.piercenet.com/method/sample-preparation-mass-spectrometry

Solid Tissue and cell culture samples Biological Fluids, Media, other sonication solution preps (i.e. IP’s) bead beater/homogenizers MW Spin filter Disruption pressure cycling technology freeze + mortar & pestle Dialysis Enzymatic Precipitation + Heat, Freeze/thaw Chaotropes(Disorder maker) Urea, thiourea, guanidine HCl, salts(KCl, NaCl, etc.), MeOH, acetonitrile Extraction Detergents Ionic (SDS), nonionic (NP40, triton x-100), zwitterionic (CHAPS) Denaturing Buffer Buffer reagent – pH considerations Buffer (denature Tris, HEPES, ammonium bicarbonate, PBS, TEAB proteins) Reducing reagents Dithiothreitol (DTT), TCEP (tris(2-carboxyethyl)phosphine) Common Extraction Buffers: 4% SDS, 100mM Tris pH 7.6, 100mM DTT 7M urea, 2M thiourea, 0.4M TEAB pH8.5, 20% acetonitrile, 4mM TCEP

Centrifuge insoluble material out & aspirate supernatant (protein extract) Depletion of high abundant proteins typically done before digestion Enrichment of post-translational modifications typically done after digestion © 2015 Regents of the University of Minnesota. All rights reserved. 64 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Disruption with the Barocycler® NEP2320 Maximizing Protein Extraction from Tissue

Pressure Cycling Technology Sample Preparation System (PCT SPS)

 Pressure Cycling Technology (PCT) uses cycles of hydrostatic pressure between ambient and ultra

35000 high levels allowing for a high degree of speed, 30000 25000

20000

reproducibility, and convenience. 15000 10000

5000 0 3:56:10 3:56:53 3:57:36 3:58:19 3:59:02 3:59:46 -5000  1 PSI input produces 440 PSI output resulting in Pressure Biosciences, Inc. a top pressure of 35 kPSI.

 Better extraction from various sample types and quicker enzymatic treatments (proteolytic cleavage, deglycosylation, etc.)

Hydrostatic Pressure Applied

Alexander Lazarev, Pressure BioSciences (2006), CHI G.O.T. Summit 2006, Boston, MA © 2015 Regents of the University of Minnesota. All rights reserved. 67 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Rapid De-pressurization Causes Membranes and Micelles to Disintegrate

Hydrostatic Pressure Rapidly Released

Alexander Lazarev, Pressure BioSciences (2006), CHI G.O.T. Summit 2006, Boston, MA

© 2015 Regents of the University of Minnesota. All rights reserved. 68 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Rat Liver Preps for 2DE, H. Ringham et al. Electrophoresis 2007, 28, 1022–1024

image: www.chemie.unihamburg.de/ bc/hahn/equipment/index_e.html

image: www.opsdiagnostics.com

PCT = pressure cycling technology, 10cycles, 20sec. 35kpsi, 20sec ambient press. GG = ground glass dounce homogenizer

500 ug loaded on each gel

© 2015 Regents of the University of Minnesota. All rights reserved. 69 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 -/+ Barocycler Prep & Digestion mouse liver tissue Treatment ABI 4800 Thermo LTQ Protein ID’s Protein ID’s

Std. Lysis/Std. Digest 115 (99%Protein Pilot) 118 (≥3peptides 99%, 203 (95%Protein Pilot) Scaffold)

Std. Lysis/PCT Digest NR 219

PCT Lysis/Std. Digest 275 264 471

PCT Lysis = 30 cycles @ 37C, 50sec. PCT Digest = 60 cycles @ 37C, Std. Lysis = mortar & pestle + sonication 35kpsi, 10sec. Ambient press. 50sec. 20kpsi, 10sec. Ambient press. Std. Digest = overnight(16hrs.)

Lysis Buffer = 8M urea, 0.2%SDS, 0.5M TEAB, 5mM TCEP Std. Digest = reduce, alkylate, dilute sample to 2M urea, add trypsin in 1:25 ratio, incubate overnight @ 37C © 2015 Regents of the University of Minnesota. All rights reserved. 70 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Biological Fluid Prep Example - Urine

MW Cut-off Dialyze Vacuum Spin Centrifuge Filter to Dryness Urine Sample Recover sample Resuspend with Solubilization buffer

Protein Concentration Assay (Bradford or BCA)

Proteolytic HPLC

Digestion or SPE Mass Spec.

© 2015 Regents of the University of Minnesota. All rights reserved. 71 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Two main sample prep paths to take...choose wisely! “When You Come to a Fork in the Road, Take It!” –Yogi Berra

http://journal.frontiersin.org/Journal/10.3389/fpls.2013.00052/full © 2015 Regents of the University of Minnesota. All rights reserved. 72 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 In-gel Proteolytic Digestion

DTT or TCEP + heat iodoacetamide (+57 mass shift on Cys) methyl methanethiosulfonate (+46 on Cys) ammonium bicarb./acetonitrile washes dry with 100% acetonitile – no speedvac 5ng/ul

Incubate trypsin digest overnight @ 37°C + trypsin, 37°C : 50/50 acetonitrile/water, 0.1% TFA or FA 75/25 acetonitrile/water, 0.1% TFA or FA pool digest and extracts for same sample R R dry down in vacuum centrifuge for SPE or mass spec. K gel cutting pictures: http://sites.psu.edu/msproteomics/category/sample-prep/in-gel-digestion-tutorial/ workflow picture: www.piercenet.com/method/sample-preparation-mass-spectrometry Adapted from: www.cshprotocols.cshlp.org © 2015 Regents of the University of Minnesota. All rights reserved. 73 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 In-Solution Proteolytic Digestion

*Protein * Peptides Extract

Dilute buffer components that interfere with proteolytic enzyme: urea < 2M, thiourea < 1M SDS < 0.1%

Digest: Add trypsin (proteolytic enzyme) in 1:35 to 1:100 enzyme to total protein ratio

Incubate overnight (12-16hrs) @ 37°C

Freeze digest at -80°C and dry down for SPE of peptides

*Consider if buffer components can be cleaned up with SPE or HPLC step before mass spec., otherwise need to rethink buffer

Reference: JR Wiśniewski, M Mann, et.al, Nature Methods 6, 359 - 362 (2009)

Adapted from www.piercenet.com

Load Proteins Buffer Digest Peptides Lysate Retained Exchange © 2015 Regents of the University of Minnesota. All rights reserved. 75 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Many different proteolytic enzymes – choose based on your application

www.promega.com © 2015 Regents of the University of Minnesota. All rights reserved. 76 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Solid Phase Extraction (SPE)/Clean-up of Peptides

Peptides + Digest Buffers Nonionic/Zwitterionic/ionic other anionic Sodium Detergents (triton-x100, CHAPS, Only Buffer & Salts hydrophobic Dodecyl NP40, SDS, etc. contaminants Sulfate (SDS)

C18 material MCX - Mixed Mode Cation Exchange

Detergent removal spin columns

Cloud Point Extraction – only for 2 punches nonionic detergents (J. Proteome Res., 2010, 9 (8), pp 3903–3911) Ethyl acetate precipitation of detergents (YG Yeung, Curr Protoc Protein Sci. Feb 2010; CHAPTER: Unit–16.12.)

cartridge images: www.perkinelmer.com image: biotage.phosdev.se ProteoSpin: http://norgenbiotek.com/display-product.php?ID=7 tips & Empore image: www.sigmaaldrich.com Pierce detergent removal: http://www.fishersci.com C18 resin image: www.lamondlab.com

Solid Tissue and cell culture samples Biological Fluids, Media, other sonication solution preps (i.e. IP’s) bead beater/homogenizers MW Spin filter Disruption pressure cycling technology freeze + mortar & pestle Dialysis Enzymatic Precipitation + Heat, Freeze/thaw Chaotropes(Disorder maker) Urea, thiourea, guanidine HCl, salts(KCl, NaCl, etc.), MeOH, acetonitrile Extraction Detergents Anionic (SDS), nonionic (NP40, triton x-100), zwitterionic (CHAPS) Denaturing Buffer Buffer reagent – pH considerations Buffer (denature Tris, HEPES, ammonium bicarbonate, PBS, TEAB proteins) Reducing reagents Dithiothreitol (DTT), TCEP (tris(2-carboxyethyl)phosphine) Common Extraction Buffers: 4% SDS, 100mM Tris pH 7.6, 100mM DTT 7M urea, 2M thiourea, 0.4M TEAB pH8.5, 20% acetonitrile, 4mM TCEP

Centrifuge insoluble material out & aspirate supernatant (protein extract) Depletion of high abundant proteins typically done before digestion Enrichment of post-translational modifications typically done after digestion © 2015 Regents of the University of Minnesota. All rights reserved. 78 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Dynamic Range Problem In Samples – A Case for Depletion

Dynamic range of proteins in human plasma

Anderson N L , and Anderson N G Mol Cell Proteomics 2002;1:845-867

© 2015 Regents of the University of Minnesota. All rights reserved. 79 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Beckman Coulter IgY-12 Protein Partitioning Column

Reference: http://www.protocol-online.org/forums/index.php?app=core&module=attach§ion=attach&attach_id=889

Reference: Agilent web seminar slides

© 2015 Regents of the University of Minnesota. All rights reserved. 81 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Sigma-Aldrich Human IgY14 and SuperMix Columns

T.Shi et al., Methods (2011), doi:10.1016/j.ymeth.2011.09.001 http://www.sigmaaldrich.com/life-science/proteomics/protein-sample-preparation/protein-depletion-products/seppro-depletion- resins/seppro-igy14-system.html/

© 2015 Regents of the University of Minnesota. All rights reserved. 82 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Enrichment - Post Translational Modifications (PTM’s)

Why Enrich for PTM of interest? In most cases, stoichiometric ratio of PTM species to unmodified species is extremely low.

Jensen Nature Reviews Molecular Cell Biology 7, 391-403 (June 2006) | doi:10.1038/nrm1939

http://www.ionsource.com/Card/phos/phos.htm Reference: TE Thingholm, Proteomics 2009, 9, 1451–1468

© 2015 Regents of the University of Minnesota. All rights reserved. 84 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Acetylation Enrichment with Anti-Acetyl Lysine Antibody

Reference: KL Guan, Nature Protocols 5, 1583–1595 (2010) doi:10.1038/nprot.2010.117

Reference: piercenet.com

© 2015 Regents of the University of Minnesota. All rights reserved. 86 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 In-Solution vs. In-Gel Strategies – Dynamic Range In-Solution Prep, 11 proteins In-Gel Prep, 401 proteins

© 2015 Regents of the University of Minnesota. All rights reserved. 87 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 INTRO: Protein Quantitation Strategies at the Peptide Level by Mass Spectrometry† • Discovery-based (complex mixture) – Stable isotope incorporation • iTRAQ® * • SILAC ** – ‘Label free’ • Spectral counting • Peptide Ion Peak Intensity: XIC-based (extracted ion chromatogram) • Targeted analyses – Select peptides of interest – Internal standard for absolute quantitation † Instrument-specific capabilities required * iTRAQ® Isobaric Tag for Relative and Absolute Quantitation ** SILAC Stable isotope labeling with amino acids in cell culture © 2015 Regents of the University of Minnesota. All rights reserved. 88 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 iTRAQ® 8-Plex Reagent Chemical Structure Isobaric Tag Total mass = 305

Amine specific peptide O O reactive group (NHS) ~~~~~~~O O N-hydroxysuccinimide Reporter Group N N N N 113 –119, 121 m/z N N O O O O This will react with primary Balance Group (?) amines...need to ensure Mass 184, 186 – 192 m/z buffer components are compatible or cleaned-up!

Applied Biosystems has granted permission to use this slide. © 2015 Regents of the University of Minnesota. All rights reserved. 89 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Isobaric Tags for Relative & Absolute Quantitation (iTRAQ®) Example: Compare Relative Protein Expression Levels in Healthy vs Disease Tissues Experimental Design for 4-plex iTRAQ® Experiment

Normal Brain Normal Brain Huntington’s Huntington’s Tissue-1 Tissue-2 Disease-1 Disease-2 Obtain protein- containing sample, extract protein

50 ug 50 ug 50 ug 50 ug Reduce, alkylate Reduce, alkylate Reduce, alkylate Reduce, alkylate Cysteines Cysteines Cysteines Cysteines

Proteolytic Digestion Trypsin Trypsin Trypsin Trypsin Digest Digest Digest Digest

Label peptides with iTRAQ TAG iTRAQ TAG iTRAQ TAG iTRAQ TAG iTRAQ® Reagents 114 115 116 117

MIX

2D LC-MS/MS Tissue Images: Rosas HD et al, (2002) Neurology, 58, 695 © 2015 Regents of the University of Minnesota. All rights reserved. 90 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2D Liquid Chromatography-MS/MS

Try to break down sample complexity

nd 1st Dimension offline HPLC 2 Dimension HPLC High pH C18 RP Inline w/Mass Spec. Low pH C18 RP

Generated with XCMS by Matt Wroblewski

Y. Wang, et al., Proteomics 2011, 11, 2019-2026

© 2015 Regents of the University of Minnesota. All rights reserved. 91 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 SILAC Metabolic Labeling Experimental Workflow

http://www.piercenet.com/method/quantitative-proteomics © 2015 Regents of the University of Minnesota. All rights reserved. 92 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 SILAC Experiment: Proteome Dynamics of B. subtilis in Response to Two Nutritional Challenges growth on phosphate succinate starvation

Lyse and Combine 1:1

adapted from Soufi B et al; J. Proteome Res. 2010, 9, 3638-3646. Copyright 2010 ACS © 2015 Regents of the University of Minnesota. All rights reserved. 93 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Label Free and Targeted Quantification Prep

Targeted

adapted from http://www.piercenet.com/method/quantitative-proteomics © 2015 Regents of the University of Minnesota. All rights reserved. 94 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Tuesday Workshop A. Stage-tip and preparation for MALDI-TOF analysis

Todd Markowski

C18 Stage Tip Clean-Up

I. Purpose A. The purpose of this SOP is to outline the assembly and usage of C18 Stop And Go Extraction Tips (Stage Tips).

II. Equipment, Reagents, and Supplies A. Equipment 1. Vortexer 2. Microentrifuge 3. SpeedVac B. Reagents 1. Water 2. Acetonitrile 3. Trifluoroacetic acid (TFA) C. Supplies 1. 1.5 mL snap-cap microfuge tubes 2. 2.0 mL snap-cap microfuge tubes 3. Hamilton syringe 4. Glass reagent bottle 5. Microfuge tube rack 6. Micropipet 7. Pipet tips 8. Empore reversed-phase extraction disk 9. 17 or 18 gauge blunt ended syringe needle 10. 1/32” outer diameter PEEK tubing

III. Hazards A. Chemical Compatibility 1. Before use of chemicals, check SDS for hazards, storage, handling, and disposing of chemicals B. Waste Management 1. Empty all liquid waste and excess reagents into the appropriate liquid hazardous waste containers a) Follow the current version of the liquid waste management and disposal SOP

Center for Mass Spectrometry and Proteomics | University of Minnesota C18 Stage Tip Clean-Up | 2

2. Place all solid biohazardous materials in appropriate solid biohazard waste containers a) Follow the current version of the solid waste management and disposal SOP

IV. Procedure A. Preparation of Reagents 1. Wash Solvent: 98:2:0.1%, water:acetonitrile:TFA a) Add 97.9 mL water to a glass reagent bottle b) Add 2 mL acetonitrile c) Add 100 µL TFA using a dedicated, gas-tight Hamilton syringe d) Mix e) Clean Hamilton syringe properly i. Rinse 3 x with water ii. Rinse 3 x with methanol

2. Wetting Solvent: 80:20:0.1%, acetonitrile:water:TFA a) Add 79.9 mL acetonitrile to a glass reagent bottle b) Add 20 mL water c) Add 100 µL TFA using a dedicated, gas-tight Hamilton syringe d) Mix e) Clean Hamilton syringe properly i. Rinse 3 x with water ii. Rinse 3 x with methanol

3. Elution Solvent: 60:40:0.1%, acetonitrile:water:TFA a) Add 60 mL acetonitrile to a glass reagent bottle b) Add 40 mL water c) Add 100 µL TFA using a dedicated, gas-tight Hamilton syringe d) Mix e) Clean Hamilton syringe properly i. Rinse 3 x with water ii. Rinse 3 x with methanol

Center for Mass Spectrometry and Proteomics | University of Minnesota C18 Stage Tip Clean-Up | 3

B. Preparation of Stage Tips 1. Place Empore disk on a flat, clean, hard surface a) A glass microscope slide is suggested 2. Press the 17 or 18 gauge blunt ended syringe needle into the Empore disk to core out a piece of the filter material 3. Press the syringe needle onto the Empore disk again for a second piece of filter material 4. Place the needle into a 200 µL pipet tip 5. Push the cored pieces into the pipet tip with PEEK tubing 6. Gently pack the material into the end of the pipet tip a) A gap of several millimeters should be visible between the disk and the end of the tip 7. Do not over pack or under pack a) Over or under packing will case the solvent to flow at a non-optimum rate and the clean-up will fail 8. The approximate binding capacity per core is 2 – 4 µg

C. Preparation of Stage Tip Assembly 1. Cut a cap from a 1.5 mL snap-cap tube 2. Bore a hole into the center of the cap 3. Snap the cap onto a 2 mL snap-cap tube 4. Place a Stage Tip into the hole of the cap a) The tip of the pipet tip should be about 1 cm from the bottom of the tube b) Alterations of the hole can be done, if necessary 5. Prepare 1 tube assembly per sample

D. Performance of Assay 1. Reconstitute sample in 60 µL Wash Solvent 2. Vortex 45 seconds 3. Centrifuge at 3000 x g for 1 minute 4. Place in 4°C until step IV.D.12 5. Place Stage Tip Assemblies in centrifuge in a balanced manner 6. Add 60 µL Wetting Solvent into the Stage Tip 7. Centrifuge at 450 x g for 2.25 minutes 8. Check to make sure all solvent passed through the membrane a) If solvent is still in the Stage Tip, use a syringe to push solvent through

Center for Mass Spectrometry and Proteomics | University of Minnesota C18 Stage Tip Clean-Up | 4

9. Add 60 µL Wash Solvent into the Stage Tip 10. Centrifuge at 450 x g for 2.25 minutes 11. Check to make sure all solvent passed through the membrane a) If solvent is still in the Stage Tip, use a syringe to push solvent through 12. Remove reconstituted sample from the fridge 13. Add sample into the Stage Tip 14. Check to make sure all sample passed through the membrane a) If sample is still in the Stage Tip, use a syringe to push it through 15. Add 60 µL Wash Solvent into the Stage Tip 16. Centrifuge at 450 x g for 2.25 minutes 17. Check to make sure all solvent passed through the membrane a) If solvent is still in the Stage Tip, use a syringe to push solvent through 18. Repeat steps IV.D15-17a once, for a total of 2 washes 19. Remove assembly from centrifuge, snap off cap with the Stage Tip, and place it on a 1.5 mL snap-cap tube 20. Place the new Stage Tip Assembly into the centrifuge 21. Add 60 µL Elution Solvent into the Stage Tip 22. Centrifuge at 450 x g for 2.25 minutes 23. Check to make sure all solvent passed through the membrane a) If solvent is still in the Stage Tip, use a syringe to push solvent through 24. Remove Stage Tip Assembly 25. Save Stage Tip in a labeled pipet rack 26. SpeedVac eluate down just to dryness

V. Maintenance A. Store all reagents, standards, and samples accordingly B. Maintenance for all equipment (SpeedVac, centrifuge, vortexer, ect) should be kept up according to lab standards

VI. Supplemental Material A. Stop and Go Extraction Tips for Matrix-Assisted Laser Desorption/Ionization, Nanoelectrospray, and LC/MS Sample Pretreatment in Proteomics Juri Rappsilber,†, Yasushi Ishihama,†,‡ and, and Matthias Mann* Analytical

Center for Mass Spectrometry and Proteomics | University of Minnesota C18 Stage Tip Clean-Up | 5

Chemistry 2003 75 (3), 663-670 DOI: 10.1021/ac026117i

VII. Contacts A. Department of Biochemistry, Molecular Biology, and Biophysics Center for Mass Spectrometry and Proteomics 46 Gortner Lab, St. Paul, MN, 55108 612-625-6283

Center for Mass Spectrometry and Proteomics | University of Minnesota Protein Estimation Methods Stephen Harvey 2019

Plasma 60 to 80mg/mL CSF 0.15 to 0.6mg/mL Urine 0 to 0.8mg/mL healthy 2.O to 20mg/mL mild disease >100mg/mL chronic kidney disease BALF <100µg/mL

Mass Spectrometry

• MALDI (Matrix-Assisted Laser Desorption Ionization) and Q-TOF (Quadrupole-Time of Flight) 60,000 Da protein requires 1.0mg/mL

• Triple Quadrupole <10 pg on column (less than ng/mL)

• ITRAQ – (isobaric tags for relative and absolute quantification) - OrbiTrap Requires 40 µg for each aliquot © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Dynamic range and Variability of proteins

Plasma Serum albumin (55% of all)

Interleukin 6, 0 to 5 pg/mL

Urine Uromodulin (30% n-glycosylated)

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Trypsin digest of a protein mfreeman_imani006_20170303_Freeman_MS... 03/03/17 10:53:40

RT: 5.60 - 10.89 NL: 6.67E7 120 Liquid chromatographic separation of peptides m/z= 100.00-4035.00 F: 7.57 FTMS + p ESI Full ms 100 [600.0000-2000.0000] MS mfreeman_imani006_201703 80 03_Freeman_MSMS_BGal_lis t 8.57 60 7.05 7.47 8.83 8.84 40 8.22 8.99 8.28 Relative Abundance Relative 7.97 9.56 5.96 6.62 9.11 20 8.08 9.32 6.79 7.70 8.66 9.22 9.48 10.21 5.71 7.10 7.14 7.39 7.87 8.49 9.71 9.80 10.71 5.91 6.01 6.18 6.25 6.40 6.48 6.67 6.85 6.98 9.84 9.88 10.02 10.25 10.43 10.52 10.76 0 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 Time (min)

RT: 5.59 - 10.90 NL: 6.47E6 120 m/z= 714.83260-714.86120 F: FTMS + p ESI Full ms 7.45 100 [600.0000-2000.0000] MS Peptide selected for fragmentation, 714.8469 m/z, mfreeman_imani006_2017030 3_Freeman_MSMS_BGal_list 80 +2 charge 60

40 Relative Abundance Relative

6.61 7.05 7.23 7.41 7.53 7.61 7.71 7.91 8.07 8.82 9.00 9.08 9.25 9.44 9.49 10.40 10.52 10.59 0 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 10.8 Time (min)

mfreeman_imani006_20170303_Freeman_MSMS_BGal_list #1967 RT: 7.46 AV: 1 NL: 1.21E6 T: FTMS + p ESI d Full ms2 [email protected] [100.0000-1480.0000] 714.84613 100 884.49329 Fragmentation mass spectra 80

60 302.11328

631.35156 40 274.11816 431.15540 545.19873 Val 698.32483 Asn 998.53693 Relative Abundance Relative 442.75024 20 614.32758 Gly Pro Glu 1127.58118 130.06511 787.44226 175.11876 244.09238 314.09793 402.24451 530.30310 564.29370 662.81927 730.41895 483.25272 867.47150 899.39014 981.50641 1027.44800 1110.55188 1140.52734 1195.57446 1254.57007 1324.63464 0 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 m/z

• Concentrate/dilute sample • Purification • Buffers and high salt • Detergents for integral protein • Immunoprecipation (IP) • Detergents throughout procedure • Gel electrophoresis

Lysis buffers - Tris, SDS, Triton x-100, EDTA

Dilution buffer - Tris, SDS, Triton x-100, EDTA

Wash buffers - Tris, SDS, Triton x-100, EDTA

Elution buffer - Tris, SDS, DTT

• Ultraviolet absorption

• Colorimetric assays Bradford Bicinchoninic Acid assay (BCA)

• Proteins Absorbance at 280nm: Tryptophan, Tyrosine Absorbance at 205nm: Peptide bond, 20-fold more sensitive, more interference • Peptides Absorbance between 200-230nm: Aromatic A.A., histidine, cysteine, methionine

Over 10-fold range in UV absorption at 280nm Extinction coeff: e = ABS/concentration (L) Varies with pH and ionic strength

Strongly interfering compounds. DNA Buffers - especially at 205nm Detergents - especially at 205nm Unsaturated compounds - 205nm

Strongly interfering compounds - DNA

DNA_ABS maxima

Protein absorbance maxima

• Nucleic acids - measure at 260 and 280nm If OD260/OD280 < 1.0, ~95% protein

• [protein] = 1.55A280 – 0.76A260 (ref. below)

• Loss to cuvette walls Dilute solutions measured at 205nm

Stoscheck, C, Methods in Enzymology, Vol 182, pg 54-56 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Colorimetric Assays

Bicinchoninic Acid assay (BCA) • 20 to 2000µg/mL • Cu2+ complexes with C, W, Y and peptide bonds • Reduced to Cu+ • Cu+ reacts with BCA reagent: • Absorbance at 562nm Coomassie Blue Protein Assay (Bradford) • 1.25 to 1400µg/mL • Basic and aromatic residues stabilizes anionic form of dye. • Peptides of over 10 residues • Absorbance at 595nm © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

Considerations

• Determine linear range. • Use similar standard to sample • Determine concentration from standard curve • Bradford - High protein to protein variability Sensitive to interfering compounds • BCA is less sensitive to interfering compound. • Microassays using these reagents are particularly sensitive to interfering agent (up 40-fold more).

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Bradford (BioRad) and BCA (Pierce) standard curves 1.0mg/mL bovine serum albumin used as standard

Bradford assay BCA assay

1 to 4µg/mL 5 to 20µg/mL

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Uromodulin concentration measure 1.0mg/mL BSA used as standard Assay Conc .(point) Conc. (slope)

Bradford(low) not detected ND (high) 0.05mg/mL ND

BCA (low) 0.17mg/mL 0.18mg/mL (high) 0.64mg/mL NA

UV280nm(high) 0.75mg/mL NA

UV280nm(Standard) 1.02mg/mL NA

Compound Bradford BCA 280nm/205nm Triton X-100 0.1% 1.0% 0.02%/<0.01% Tween 20 0.01% 1.0% 0.3%/0.1% CHAPS 10% 1.0% 1.0%/1.0% SDS 0.1% 1.0% 0.1%/0.1% Octyl-B-glucoside 0.5% 5.0% 10%/ND Tris 2.0 M 0.1 M 0.5 M/40mM Phosphate 1.0 M 2 M 1.0 M/50mM Sodium chloride 5.0 M 1.0 M >1.0 M/0.6 M

Compound Bradford BCA LC-MS Triton X-100 0.1% 1.0% not Tween 20 0.01% 1.0% not CHAPS 10% 1.0% SDS-PAGE SDS 0.1% 1.0% 0.1%SDS-PAGE Octyl-B-glucoside 0.5% 5.0% yes Tris 2.0 M 0.1 M not Phosphate 1.0 M 2 M not Sodium chloride 5.0 M 1.0 M not (10uM – ion suppression)

• Complicate interpretation

• Compete with charged ions

• Interfere with MALDI matrix

• Signal suppression

• Contaminate the instrument

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Example contaminant affects on mass spectrometry analysis m/z (H+) 854.3382 SALTS • Complicate interpretation

McTavish_20150308_sample2_PAX #1 RT: 0.00 AV: 1 NL: 2.97E7 T: FTMS + p ESI Full ms [150.00-2000.00] 854.33990 100

80 +H 70

60 10mg/mL in methanol 855.34308 50 ~10,000ppm

40 Relative Abundance 30

20 +Na +K +2Na 856.34613 892.29700 899.39789 876.32074 10 893.29816 857.34955 877.32672 894.30225 885.37683 0 840 845 850 855 860 865 870 875 880 885 890 895 900 m/z

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 McTavish_20150308_1_100_PAX_150310152526 #1 RT: 0.00 AV: 1 NL: 4.25E8 T: FTMS + p ESI Full ms [150.00-2000.00] 876.32233 100 +Na 0.1mg/mL 90 Diluted in Sigma Optima grade 80 892.29578 Methanol >2ppm sodium 70 >1ppm potassium 60 +K 877.32526 50

40 +H 893.29919

Relative Abundance 854.34021 30 ~100ppm

20 855.34375 894.29974 878.32837

10 856.34650 873.81531 895.30017 879.33136 846.56982 853.56433 857.34912 865.82996 871.36804 884.30920 896.30414 0 845 850 855 860 865 870 875 880 885 890 895 900 m/z McTavish_201503011_MeOH_1_10000B_PAX #1 RT: 0.00 AV: 1 NL: 1.22E8 T: FTMS + p ESI Full ms [150.00-2000.00] 876.32172 100

90 +Na 0.001mg/mL

70 ~1.0ppm

50 877.32501 +K 892.29535 40 Relative Abundance 30 893.29895 20 +H 878.32825 894.29980 10 854.33990 858.71478 879.33240 895.30017 841.72809 851.70947 865.62347 869.70319 873.71393 886.74792 0 840 845 850 855 860 865 870 875 880 885 890 895 900 m/z © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Fragmentation of compound.

McTavish_20150308_MSMS_1_100_PAX #1 RT: 0.00 AV: 1 NL: 2.01E8 T: FTMS + p ESI Full ms2 [email protected] [150.00-2000.00] 308.09 100

90 (285.1061, +Na) 80

60 (568.237, +Na) (853.336, +Na) 591.22 876.32 50

Relative Abundance (508.216, +Na)

30 531.20 587.55 20 398.24 892.30 10 509.22 609.16 865.63 281.15 327.16 559.52 165.07 240.10 387.18 409.16 469.18 625.13 710.23 832.27 685.44 770.25 909.66 953.68 998.38 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 m/z

• Expected fragment peaks are present complexed with sodium (+23,).

• Salt adducts may produce different ion signal response.

• Salt adduct are present with fragmentation. Complicating identification.

McTavish_20150313_60ng_uLPL_1000pg_uLPAX #621-841 RT: 1.44-1.95 AV: 221 NL: 8.09E7 T: FTMS + p ESI Full ms [150.00-2000.00] +4 125 120 115 McTavish_20150313_60ng_uLPL_1000pg_uLPAX #1 RT: 0.00 AV: 1 NL: 1.92E8 + 110 Compound w/Na T: FTMS + p ESI Full ms [150.00-2000.00] 105 900.87019 714.42 100 100 886.19502 692.41 95 871.51908 90 95 856.84363 736.43 85 80 901.87067 90 681.40 75 885.15996 887.19683 899.83536 747.44 70 855.80914 872.52118 85 65 857.75600 60 55

80 Relative Abundance 50 75 45 670.39 40 70 35 30 876.32010 758.45 25 659.39 65 20 15 858.50942 868.51132 877.32187 888.02197 890.52063 904.18672 60 769.45 10 880.26982 862.16660 866.51451 906.19097 5 853.75648 +3 864.19145 874.50116 893.54071 850.48979 883.51094 895.86616 55 0 850 855 860 865 870 875 880 885 890 895 900 905 780.46 930.22 m/z 50 886.19 915.54 45 648.38 813.48 871.52 944.90 Relative Abundance 40 +5 974.25 856.84 35 637.77 827.49 1003.93 30 1018.61 620.16 25 1032.95 611.35 20 1047.62 15 602.75 1077.31 +2 10 1091.99 1273.77 1339.81 585.14 1229.74 1405.85 5 567.33 1150.69 1449.88

0 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 1450 m/z

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Detergents • Signal suppression • Contaminate the instrument

octylglucoside octylglucoside Before cleanup After cleanup

Triton X-100 After cleanup

Yeung,Y, Nieves,E, Angeletti, R, Stanley, E Anal Biochem. 2008 Nov. 15; 382(2): 135-137 © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Polyethylene Glycol and detergents

• Complicate interpretation

• Compete with charged ions

• Interfere with MALDI matrix

• Signal suppression

• Contaminate the instrument

Ethylene oxide polymer ~44 m/z

Develop purification/enrichment techniques with both protein level assay and mass spectrometry compatibility in mind.

• Ultraviolet spectroscopy 280nm proteins: 200 - 230nm proteins or peptides Differential absorption at 280nm, determine extinction coefficient Sensitive measure at 205nm, many interfering compounds

• Colorimetric assays Dye-based, Bradford Cu+2 based, BCA Variable protein-to-protein sensitivity Interfering compounds • Mass spectrometry picogram to microgram quantities required Signal suppression Detergents Non volatile salts Etc. © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Compatible concentrations for protein assays

Compound Bradford BCA UV280nm/205nm Acetate 0.6 M 0.2M 0.1/0.01 Acetone 10% 10% Ammonium sulfate 1.0 M 1.5M 50%/9% Ampholytes pH 3–10 0.5% BES 2.5 M Brij 35 5.0% 1%/1% CHAPS 10% 1.0% 1%/1% Citrate, 50 mM <1.0mM 5%/<10mM Deoxycholate 0.1% 5% 0.3%/0.1% DNA/RNA 10.25mg 0.1% 0.1mg/1.0µg DMSO 10% 10% 20%/<10% DTT 1.0 M <1mM 3mM/0.1mM

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Compound Bradford BCA UV280nm/205nm EDTA 0.2 M 10mM 30mM/0.2mM EGTA 50 mM Glycerol 99% 10% 40%/5% Glycine 0.1 M 1.0M 1.0M/5.0mM Guanidine-HCl 2.0 M 4.0M HEPES 0.1 M 0.1M ND/<20mM Imidazole 200mM 50mM β-Mercaptoethanol, 1.0 M 0.01% 10mM/<10mM MES 0.7 M 0.1 M Methanol 10% 10% MOPS 0.2 M 0.1mM NP-40 0.25% 5.0% Octyl-B-glucoside 0.5% 5.0% 10%/ND OTG 1.0% 5.0%

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Compound Bradford BCA UV280nm/205nm Phosphate 1.0 M 2 M 1.0 M/50mM PIPES 0.5 M 0.5 M Potassium chloride 1.0 M <10mM 100mM/50mM SDS 0.1% 1.0% 0.1%/0.1% Sodium azide 0.5% 0.2% Sodium chloride 5.0 M 1.0 M >1.0 M/0.6 M Sucrose 1.0 M 40% 2.0 M//0.5 M Tris 2.0 M 0.25 M 0.5 M/40mM Triton X-100 0.1% 1.0% 0.02%/<0.01% Tween 20 0.01% 1.0% 0.3%/0.1% Urea 6.0 M 3.0 M >1.0 M/<0.1 M

Stoscheck, C, Methods in Enzymology, Vol 182, pg 52-53 Noble, J.E., Methods in Enzymology, Vol 463, pg 73-95 Pierce, ref 1296.2 BioRad Laboratories Inc, technical note 1069

PPS Silent Surfactant http://shop.expedeon.com/products/18-Protein-Solubility/ 129-PPS-Silent-Surfactant/ Big CHAPS deoxy http://www.emdmillipore.com/life-science-research/big-chap-deoxy/ EMD_BIO-256455/p_Ltab.s1L_.8AAAEWhmEfVhTm?PortalCatalogID= merck4biosciences&CountryName=United+States+of+America ASB series http://www.emdmillipore.com/life-science-research/asb-16 /EMD_BIO-182755/p_hmmb.s1L_.4AAAEWhmEfVhTm RapiGest SF http://wwwp1.waters.com/webassets/cms/support/docs/715000122.pdf ProteaseMax http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20 Bulletins/101/ProteaseMAX%20Surfactant%20Trypsin%20Enhancer.pdf N-octyl-B-glucopyranoside Sodium Deoxycholate

Trition X-100

NP-40

Igepal

Bri-35

Tween-20

Octyl-B-thioglucopyranoside

SDS*

CHAPS*

* May be removed by SDS-PAGE

Tuesday Workshop C. Sample preparation technologies and instrumentation

Bruce Witthuhn

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 General Strategy for Protein Analysis cell pellet or crude tissue

1. Disrupt cells (freeze thaw or grinding) 2. Add extraction buffer 3. Centrifuge and collect supernatant

Soluble extract of total cellular protein Measure protein concentration (buffers must be compatible!!) Label with Cy3 or Cy5 •Mass spectrometry •2D HPLC separation of Intact proteins DIGE--quantitate •Column chromatography •Native gel electrophoresis •ELISA In gel trypsin digest •Bioassays •ICAT or iTRAQ Protein ID by Protein ID by Mass Spectrometry

Mass Spectrometry 133 Sample Prep

• Protein or cell quantitation is an important step in sample preparation  to perform differential analysis an equal number of cells or equivalent amounts of total protein must be compared • Cells can be counted prior to lysis

• Protein content can be determined by spectrophotometry after lysis (dye binding)  not absolute, but relative  compared against a known standard

• Sample prep  must be optimized  key to successful study

134 2D Gel Electrophoresis

Complex mixture of proteins

Denature and solubilize in solution (Sample prep)

Separate proteins by charge in first dimension (IEF)

Separate proteins by size in second dimension (SDS-PAGE)

Individual proteins isolated as distinct protein features within a gel matrix

135 Typical Results Following 2DE

200 kDa

10 kDa pH 4 pH 7

136 Visualization of results

Most detection methods used for SDS gels can be applied to second-dimension gels.

• High sensitivy • Wide linear range for quantification • Compatibility with mass spectrometry

Autoradiography and fluorography: 200 fg protein (film vs storm imaging 5 orders of magnitude)

Silver: (visible) below 1 ng linear range 1 – 60ng

Coomassie: (visible) 100 ng/protein spot

Sypro Ruby: (flourecences) 1ng linear range 1-1000ng

DIGE: (flourescences) 0.125ng linear range 0.125ng - 10 ug

ProteoSep; 214nm 500ng (3x signal to noise)

MS detection limits ~ 500fm 50 kD protein 100fm/5ng

137 Differential In Gel Electrophoresis

138 Differential In Gel Electrophoresis (DIGE)

Tissue or cells are harvested and placed in lysis buffer ( 30mM Tris (pH8.5), 7M urea, 2M thiourea and 4% (w/v) CHAPS) at 1mg/ml on ice.

Samples are cleared of insoluble material by centrifugation (100,000g).

Samples are quantitated and brought to a final concentration of 5ug/ul.

Proteins are then subjected to limited labeling as follows. 1ul of 400uM cy3 or cy5 in dimethyl formamide is added to 10 ul of 5ug/ul protein lysate. Samples are incubated on ice for 30’ and reaction is stopped by adding 1ul of 10mM lysine.

Samples are combined in 250ul final volume of immobilized pH gradient (IPG) running buffer (7M urea, 2M thiourea, 10mM dithiothreitol, 0.5% IPG buffer ( same pH range as strip)) and allowed to rehydrate the IPG strip overnight under low current (30V for 7 hrs followed by 60 V for 7 hrs). After overnight rehydration the samples are resolved with a voltage ramp of 500V/1hr, 1000V/1hr and 8000V/2hrs in the Amersham Ettan™ IPGphor ™IEF until a total 17500 VHrs is reached.

Strips are equilibrated for 0.5-1hr in SDS equilibration buffer (50mM tris pH 8.8, 8M urea, 30% glycerol, 4%SDS) followed by resolution on a SDS-PAGE.

Gels are visualized utilizing the Typhoon 8600 with the following settings (Cy3: Excitation 532nm, Emission filter 580BP30, Cy5: Excitation 633nm, Emission filter 670BP30).

Differences between samples are analyzed using the DeCyder software program.

Proteins spots of interest will be selected from 2D gels loaded with 100-500ug of sample.

Proteins will be aseptically robotically excised from the gels using the Genomic Solution™ Investigator ProPic™ .

Selected spots will be subjected to enzymatic digestion, C18 micro purification and spot dispensing on MALDI-TOF targets on the Genomic Solution™ ProPrep™ .

139 CyDye™ DIGE Fluor Minimal Dyes

The NHS-ester reactive group of CyDye™ covalently attaches to the epsilon amino group of lysine of proteins via an amide linkage.

3% of the available proteins are labeled on a single lysine per protein.

CyDyes have been selected to not alter the net charge of the lysine at neutral PI and contribute identical MW changes ensuring that the pI and MW of the protein does not significantly alter compared with the same unlabelled protein.

CyDye™ DIGE Fluor Labeling Kits for Scarce Samples and Preparative gels

The maleimide reactive group of the saturation dyes covalently bonds to the thiol group of cysteine residues of proteins via a thioether linkage.

To achieve maximal labeling of cysteine residues, the protocol uses a high fluor to protein labeling ratio. They have a net neutral charge and therefore do not alter the charge on a protein after labeling.

They are also matched for molecular weight ensuring that the same protein from a standard and test sample labeled with the different dyes will overlay when separated on a single gel. 140 Typical Results Following 2DE

200 kDa

10 kDa pH 4 pH 7

141 Typhoon 8600 Scanned Gels

Cy5 Cy3 pH4 pH 7 pH4 pH 7

142 Spot Picking

143 Normalization

• Calculate all spot volumes • Calculate ratio of spot volumes between gels • Assumed most spots don’t change optimize ratios of spots around zero. • Factor all spots on primary gel to be equivalent to secondary gel. • Can be extended to groups of gels

144 145 DIGE Graph

146 DIGE 3D Volume

147 Additional Treatments

B. Acetone precipitation Sample loss may be as high as 25% a. occasionally not all of the interfering substances removed b. difficult to get proteins back into solution *I use ethanol instead

C. ProteoSpinTM Detergent Clean-up Kit form Norgen Biotek Corp Provides a fast and effective procedure to remove detergents including SDS, Triton® X-100, CHAPS, NP-40 and Tween 20.

D. Thermo Scientific and SIGMA deteregent removal spin columns now available

148 4-Plex iTRAQ Reagents

Amine Specific Labeling on Lysines and N-terminus

Label up to 4 samples

Labeling of peptides shown

Ross etal 2004, Molecular and Cellular Proteomics, 3, 1154-1169

149 iTRAQ (Amine-specific Labeling) with Tandem Mass Spectrometry

150 Strong Cation Exchange Chromatography - MV sample

UV2 022112MayankVerma_11353_MDXG8plex_SCX 40 40

35 35

30 30

25 25 mAU mAU 20 20

15 15

10 10

5 5

0 0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 Minutes

151 High pH RP Chromatography - MV sample

UV2 25 022311_MayankVerma_11353_HighpH 25

20 20

15 15 mAU mAU

10 10

5 5

0 0

0 10 20 30 40 50 60 70 80 90 Minutes

152 Total Ion Chromatogram from 2nd Dimension of one fraction

153 2 types of spectra

full scan shows the mw of the peptides ms/ms shows the mw for ions corresponding to the amino acid sequence

154 Glu-Fib peptide 8plex

1874.8842

1873.8741

1864.8 1871.6 1878.4 1885.2 MS/MS Mass (m/z)

y9(+1) 100 1.1E+4 90

50 y6(+1)

% % Intensity 40 116.1147 y13(+1) 30 y1(+1) b2(+1) y7(+1) 20 b3(+1) b6(+1) y2(+1) y8(+1) 1039.4237 10 V y4(+1) b4(+1) b5(+1) 1268.5028 b14(+1) y15(+1) E R 344.1307 y14(+1) 188.1420 565.6216 795.3037 a8(+1) 1163.4664 1423.7990 0 9.0 403.4 797.8 1192.2 1586.6 1981.0 Mass (m/z) Reporter ions

116 116 121 A. 113 114 115 117118 119 121 B. 117 F 120.10(A14747.87) F R 114 115 118 120.08 112.10(A9043.70) R 113 119

111.04(A162.86) 123.03(A199.04) 113 116 119 122 114 117 120 123 Mass (m/z) Mass (m/z) Expected Ratio: Expected Ratio: 1:1:1:1:1:1:1:1 1:2:2:5:5:2:1:5

155 MS/MS Spectrum

y1 291.21 VSFLSALEEYTK

[M +2H] 2+ 837.02 b1 b3 c 244.18 478.28 p y3 b4 y2 555.33 591.36 s b2 331.21 392.26 y8 y7 1084.56 y4 y5 b5 b6 997.53 749.38 y6 m/z residue B y V 244.18 1674.92

S 331.21 1431.75 F 478.28 1344.72 L 591.36 1197.65 S 678.39 1084.56 A 749.43 997.53 L 862.52 926.50 E 991.56 813.41 E 1120.60 684.37 Y 1283.66 555.33 T 1384.71 392.26 156 K 1656.91 291.21 Protein Digestion

Washing: A series of water, ammonium biocarbonate and acetonitrile additions and aspirations to remove impurites from the separation

Reduction:

Alkylation:

Washing and Dry:

Digestion: Trypsin 12ngul, 25-100mM ammonium bicarbonate other enzymes, AspN, GluC, Chymotrypsin

Extraction of peptides

Zip Tip: Concentration/desalting

Mass spectrometry Identification

157 Reduction

BME HS-CH2CH2-OH DTT HS –CH2 (CHOH)2 CH2- SH [ Tri-(2-carboxyethyl) phosphine]

R-SS-R + HS –CH2 (CHOH)2 CH2- SH R-SH + R- SS –CH2 (CHOH)2 CH2- SH

R-SH + R-SS –CH2 (CHOH)2 CH2- SH R-SH + S-S

CH2 (CHOH)2 CH2

Alkylation

IAA and its analogue IAM are the classical and widely used reagents for alkylation of cysteines (methyl methanethiosulfonate(MMTS)

R-CH2SH + ICH2CONH R-CH2SCH2CONH + I-

103.1 103.1 + 57 158

Wednesday AM Lecture 1: Mass Spectrometry Instrumentation

Tom Krick

Mass spectrometry is a powerful analytical technique that is used to identify unknown compounds, to quantify known compounds, and to elucidate the structure and chemical properties of molecules.

• Identify structures of biomolecules, such as carbohydrates, nucleic acids and steriods • Sequence biopolymers such as proteins and oligosaccharides • Determine how drugs are used by the body • Perform forensic analyses such as conformation and quantitation of drugs of abuse • Analyze for environmental pollutants • Determine the age and origins of specimens in geochemistry and archaeology • Identify and quantitate compounds of complex organic mixtures • Perform ultrasensitive multielement inorganic analyses

(Ref: http://www.asms.org/whatisms)

159 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Goals Attainable by MS

• Small molecule MW & structural composition • Intact protein molecular weight • Peptide mass • Peptide sequence  protein ID • Identification and location of post-translational modifications of amino acids • de novo Sequencing of unknown proteins • Relative Quantification of Proteins

160 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 History

1803 John Dalton proposes theory that each element has distinct, measurable atomic weight*

1905 J. J. Thomson produces the first mass spectrum (e/m) (Cambridge University)*

1919 Francis Aston discovers elements have stable isotopes (Cambridge University)*

2002 John Fenn (Virginia Commonwealth) and Koichi Tanaka (Shimadzu Corp., Japan) joint Chemistry Nobel Prize winners for ionization techniques ES and MALD

* Source: Measuring Mass: from positive rays to proteins; Grayson MS, ed. Chemical Heritage Press 2002

161 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 How does a mass spectrometer work?

• Ion Source: Makes ions • Mass Analyzer: Separates Ions • Detector: Presents information as a Mass Spectrum

So for a mass spectrometer to work, the molecules must be in the gas phase and ionized so they can be mass separated

162 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Mass Spec Principles

Sample

+ _

Ionizer Mass Analyzer Detector

Figure: Dr. David Wishart, University of Alberta

163 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Electron Impact (EI) and Chemical Ionization (CI) Spectra of Ephedrine

Reference: asms.org

164 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Mass Spectrometry Data: m/z

• Mass (m): • calibrate instrument with standard compound (molecule with a known MW) • Charge (z): • Calculate CHARGE STATE OF AN ION from peak spacing in RESOLVED ISOTOPE SERIES/ENVELOPE • MALDI mainly produces singly charged ions - easy to interpret +TOF MS: Experiment 1, 0.16... Max. 6140.0 counts. +TOF MS: 78 MCASINGLY scans from... CHARGEDMax. 707.0 counts. a=3.57102922947957410e-00...DOUBLY CHARGED a=3.56297358347516750e-004... 482.276 1438.736 3000 662 1439.734

600 ts n u o c , ity s n te In

nt t ts n u o c , ity s n te In 2500 500 1 2000 400 482.777 1500 300 1440.737 1000 Intensity, cps cps Intensity, 200 483.281 0.5 100 500 0 1438 1440 1442 cps Intensity, 0 m/z, amu 482.0 482.5 483.0 483.5 484.0 m/z, amu

165 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 http://www.ionsource.com/Card/Mass/mass.htm

166 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peaks in an Isotope Series have Identical Structural Formulas but Different Isotope Components +TOF MS: Experiment 1, 46.580 min from keyxx001_perez061_fxn19_queb2_1004.wiff Max. 2754.0 counts. a=3.56649482550429800e-004, t0=4.09124284567078580e+001, Smoothed 696.70 895 696.36 800

600

697.03 400 Intensity, counts counts Intensity,

Intensity, counts 200 697.36

0 696 698 m/z 12C, 14N, 16O, etc – containing ions m/z, amu13C, 15N, 18O, etc – containing ions

167 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Mass Spectrometry: Resolution Monoisotopic vs. Average m/z

“Mass resolution is the dimensionless ratio of the mass of the peak divided by its width. Usually, the peak width is taken as the full width at half maximum intensity, (FWHM).”

Average peak Monoisotopic peak Reference: http://cws.msi.umn.edu/mascot/help/mass_accuracy_help.html

168 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 The Resolving Power of a Mass Spectrometer Dictates the Accuracy of the m/z Values Produced m/z Value tolerance accuracy 1000 1 1000 ppm

1000.3 0.1 100 ppm

+TOF MS: 38 MCA scans from russeth0611a_5S_fs.wiff Max. 996.0 counts. a=3.56274449646042090e-004, t0=4.13301967471197710e+001, Smoothed 995.568 1000.345 0.001 1 ppm 155 140

120

100 996.566

RESOLUTION = 8000 60

Intensity, counts40 997.555 Absolute Intensity Absolute Intensity 20

0 992 994 996 998 1000 1002 m/z, amu

169 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Mass Accuracy Calculations

Accuracy in ppm (parts per million):

∆ x 106 = ppm error theoretical value

Accuracy in Percent

∆ x 100 = % error theoretical value

∆ = experimental/observed value (mass spec data) – theoretical value

300 ppm is equivalent to 0.03 %

170 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Comparison of Resolving Power: 645.7 m/z

645.793

FT-ICR MS 646.791 (Fourier Transform-Ion Cyclotron Resonance) Absolute Intensity Absolute

645 646 647 648 m/z +TOF MS: Experiment 1, 46.580 min from keyxx001_perez061_fxn19_queb2_1004.wiff Max. 2754.0 counts. a=3.56649482550429800e-004, t0=4.09124284567078580e+001, Smoothed 645.70 247 646.04

200

150 Quadrupole-TOF MS 646.37 (Time of Flight) 100

Intensity, counts 50 646.70

0 645.0 646.0 647.0 648.0 m/z, amu

171 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MALDI-TOF: Peptide Mass Fingerprint In-Gel Tryptic Digest: GOAL = Protein Identification

1600

1400

1200

1000

800

600 Absolute Intensity Absolute

400

200

800 1000 1200 1400 1600 1800 2000 m/z

172 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 ESI-Ion Trap MS: Product Ion Spectrum

MS/MS Spectrum from [M + 2H+2], Precursor m/z = 467.8 (MW = 933)

750 x50 100

75 637

185 50

566 376 Relative Abundance Relative 25 438 789 298 864 157 253 369 497 701 0 100 200 300 400 500 600 700 800 900 m/z

173 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MALDI-TOF MS of Intact Protein: Unoxidized Calmodulin

174 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MALDI-TOF MS of Intact Protein: Mixture of Calmodulin with Multiply Oxidized Methionines a. i .

16832, 7 ox-Met

200 16817 16849, 8 ox-Met 6 ox-Met

150

100 16800 5 ox-Met E. Balog, Univ MN, BMBB

0 16600 16800 17000 m/z / i =/mar01/ cl 0306a6/ 1SLi n/ pdat a/ 1 Admini st rat or Sun Mar 11 23: 46: 53 2001

175 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Intact Protein ESI-MS: Cytochrome C +12 100 1031.0 +13 1) Raw MS data 951.8 +11 1124.6 +8 1545.7

+14 +10 884.0 1236.9 50 +9 2) Protein MW 1374.2 +7 (after mathematical deconvolution) 1766.6 Relative Abundance Relative +15 825.0 +17 +16 727.5 772.4 0 1000 2000 200 m/z 12359 +/- 2 100

Mathematical deconvolution 50 Relative Abundance Relative

0 12500 15000 10000 mass

176 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Mass Spectrometry Principles

Sample

+ _

Ionizer Mass Analyzer Detector

Figure: Dr. David Wishart, University of Alberta

177 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 IONIZATION

• Mass spectrometers can detect the presence of IONS (+ or –) • Ions must be in the GAS PHASE • Ions must be free from salts, solvents... • Ionization from SOLID CRYSTALS • Destroys non-covalent interactions • MALDI (Matrix Assisted Laser Desorption Ionization); typically produces singly charged ions • Ionization from LIQUID STATE: • ESI (Electrospray Ionization); typically produces multiply charged ions • Non-covalent interactions are retained • Compatible with in-line HPLC • solvents must be volatile- e.g. ammonium acetate, ACN, methanol • Not all ionization modes are applicable to a given molecule

178 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Electron Impact Ionization

Odd-electron ions (radical cations) are formed during Electron Impact

179 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Ionization Techniques for MS

• Electron Impact (EI) • Chemical Ionization (CI) • Inductively Coupled Plasma (ICP) • Field Desorption (FD) • Fast Atom Bombardment (FAB) • Thermospray • Desorption Electrospray Ionization (DESI) • Direct Analysis in Real Time (DART) • Atmospheric Pressure Chemical Ionization (APCI) • Secondary Ion Mass Spectrometry (SIMS) • Matrix-assisted Laser Desorption (MALDI) • Electrospray (ESI)

180 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Technology Breakthroughs (1990’s) in Mass Spectrometry Accelerated Proteomics Studies

Ionization Methods suitable for Peptides/Proteins (i.e. methods for producing gas phase ions):

• Electrospray Ionization: John Fenn (Virginia Commonwealth) 2002 Nobel prize Chemistry

• MALDI (matrix-assisted laser desorption ionization): Koichi Tanaka (Shimadzu Corp) 2002 Nobel prize Chemistry

181 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Soft Ionization Methods

337 nm UV laser Liquid Analyte

+ _

analyte + matrix needle

MALDI ESI • Unlimited analyte mass • Unlimited analyte mass • Insensitive to moderate salts • Some sample cleanup Mixtures problematic • Excellent for mixtures • • On-line sample injection • Off-line sample prep Adapted from Dr. David Wishart, University of Alberta

Reference: Prosilia Omni Spray™ Ion Source manual

185 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Instrumentation Multiple types of Mass Analyzers are used for a variety of purposes

186 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MALDI with Time-of-Flight (TOF) MS velocity of the ion in the flight tube depends on the mass-to-charge ratio

N2 Laser 337 nm Time of Flight tube Ion sensitive ion source Detector fast + slow + +

vacuum: 10-7 Torr

Target: Analyte Ions + “matrix” crystals Apply Accelerating Potential (19kV)

187 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Image source: http://www.anagnostec.de/index/modul/portal/kernwert/technology/

188 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 The Time an Ion Spends in Flight Summary: Time of flight of the ion varies with the square root of its mass-to-charge ratio m tx 2zeUex

mt RP

Time (ms) (ms) Time mt2

0 m/z (z=1) 10,000 D.C. Muddiman Chemistry 727

189 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 QUADRUPOLE Mass Filter

Reference: http://www.chm.bris.ac.uk/ms/theory/quad-massspec.html

190 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Quadrupole Mass Analyzer

• Uses a combination of RF and DC voltages to operate as a mass filter • Has four parallel metal rods with alternating positive and negative voltages • Can scan through all masses or sit at one fixed mass

191 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Comparison of Linear Quadrupole to 3-D Quadrupole (Ion Trap)

Figures from: http://www.abrf.org/ABRFNews/1996/September1996/sep96iontrap.html

192 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3D Ion Trap The ion trap is an energy well. Ions with sufficient energy to enter the trap are retained by an energy barrier on the exit side of the trap. The advantage of the ion trap is that it accumulates selected ions prior to their analysis giving it high initial sensitivity. Ions can be fragmented by collision with helium gas and their product ions analyzed within the trap. Selected product ions can undergo further fragmentation, thus allowing MSn. The ion trap has a high efficiency of transfer of fragment ions to the next stage of fragmentation (unlike the triple quadrupole instrument).

The 3D ion trap consists of two end cap electrodes and a center ring electrode

193 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 ION TRAP MASS ANALYZER SCHEMATIC

Figure from: http://www.matrixscience.com/help/ion_trap_main_help.html

194 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 HYBRID QUADRUPOLE TIME-OF-FLIGHT MASS SPECTROMETER

Reference: Chernushevich IV et al. J. Mass Spectrom. 2001; 36:

195 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 LTQ Orbitrap Velos Schematic

linear ion trap multipole

Page 1-3 LTQ Orbitrap Velos Hardware Manual, Thermo Fisher Scientific

http://en.wikipedia.org/wiki/Orbitrap

196 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

Wednesday AM Lecture 2: ESI-LCMS Peptide MSMS

LeeAnn Higgins

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Outline Terminology • Liquid Chromatography • ESI • Electrospray • Liquid chromatography • Acquisition • MS Acquisition • DDA (data dependent • Peptide Fragmentation acquisition) • PTM Detection • MS1 vs MS2 • Protein ESI-MS • Precursor ion • MS/MS • Product Ions • b-ion, y-ion

197 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 EXAMPLE Workflow: MIXTURE OF UNKNOWN PROTEINS

SDS-PAGE Gingras A et al, 2007 Nat Rev Mol Cell Biol 8, 645 A. Obtain protein-complex B. Separate proteins on 1D gel C. Excise protein bands > trypsin digestion D. Liquid Chromatography-ESI-Mass Spectrometry

198 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Example Sample Analysis Workflow

Liquid Chromatography (LC): Peptide Separation

Electrospray Ionization (ESI)

Mass Spectrometry (MS)

199 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Liquid Chromatography: Separation Technique

Mixtures are separated or partially separated before MS analysis with Chromatographic Technologies

200 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Liquid Chromatography Basic Overview

“… modern Liquid Chromatography (LC), uses a liquid mobile phase to transport the sample components through a column packed with a solid material - the stationary phase.” Reference: http://www.earl2learn.com

http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

Mikhail Tswett (1872 – 1919)

From Tswett’s notebook (1910) on the early chromatographic experiments: plant pigments were passed through calcium carbonate using petroleum ether

201 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Liquid Chromatography, Peptide Elution Profile 1D LC

i022_091711_sPRG_pep_digestmix_HC... 9/17/2011 11:27:17 PM

0.00 - 70.01 41.21 100 • Peaks represent analyte elution 26.13 36.27 profiles 90 9.43 38.80 31.80 80 • Increased Retention Time = 24.56 increased peptide hydrophobicity 70 on a C18 column 33.72 60 19.70 28.01 48.65 50 18.35 20.49 44.71 40

30.98 30

17.65 20 44.87

15.64 55.25 50.56 10 9.06 57.67 2.57 8.53 12.71 52.05 58.72 63.03 67.12 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 Time (minutes)

202 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 • Column 1 Elution Profile • Use fraction collector – collect peptides in separate tubes Liquid Chromatography, Peptide Elution Profiles 2D LC (214 (214 nm) mAU

Intensity, Intensity, Column 2: LC-MS Peptide Elution Profiles

2 minute intervals 10 20 30 40 50 60 70 Minutes Mass Spec Absolute Intensity Absolute Spec Mass

Time (sec)

203 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Example Sample Analysis Workflow

Liquid Chromatography (LC): Peptide Separation

Electrospray Ionization (ESI)

Mass Spectrometry (MS)

204 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Electrospray Ionization (ESI) Produce Analyte Ions

GOAL: eliminate solvent, get analyte into the gas phase; apply high voltage to a liquid to create aerosol

http://www.newobjective.com/electrospray/index.html

205 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Electrospray Ionization

Liquid flow Mass Spectrometer Mass

Ref: IJAC Vol 2012 ID 282574

206 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Example Sample Analysis Workflow

Liquid Chromatography (LC): Peptide Separation

Electrospray Ionization (ESI)

Mass Spectrometry (MS)

207 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 EXAMPLE Workflow: MIXTURE OF UNKNOWN PROTEINS

Typical Numbers of Proteins Identified:

• 1D LC-MS: up to 300 • 2D LCMS: up to 5000

(sample and dynamic range dependent)

208 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 EXAMPLE: MIXTURE OF UNKNOWN PROTEINS

Data-Dependent Acquisition Scan Mode (DDA) on the Mass Spectrometer

209 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 Dimensional LC-ESI-MS Elution Profile

RT: 0.67 - 73.58 Retention time 39 minutes 39.08 100 Next slide: see Full Scan/Survey Scan at 39 minutes 90

60 28.56 42.32 50 46.09 53.64 49.61 40 59.65 Relative Abundance 24.45 30 32.33 34.17 54.19 57.26 20 17.92 62.44 10 22.91 63.78 13.80 17.67 68.37 1.63 4.17 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Time (min)

210 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MS1 Data Acquisition • MS1 spectrum (below) • Peaks below represent unfragmented peptide m/z values

georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7 F: FTMS + c NSI Full ms [360.00-1800.00] 851.95 100

70 Mass Spectrum at 39.22 minutes shows co- 60 eluting peptides

50 671.68 782.40 40 553.61 587.05

Relative Abundance 829.91 30 504.01 666.34 20 689.84 10 863.44 477.43 739.37 1007.02 1173.09 616.32 929.73 445.12 1063.51 1136.26 1239.31 1350.02 1427.47 0 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

211 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Data Dependent Acquisition: • Top 6 (most abundant) Peaks Identified • Next 6 Scan Events are MS/MS georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7 F: FTMS + c NSI Full ms [360.00-1800.00] 1 851.95 100

90 Sequential MS/MS scan events 80 are triggered for the 6 most 70 abundant peaks 60 2 50 3 782.40 5 4 671.68 40 553.61 587.05 6 Relative Abundance 829.91 30 504.01 666.34 20 689.84 10 863.44 477.43 739.37 1007.02 1173.09 616.32 929.73 445.12 1063.51 1136.26 1239.31 1350.02 1427.47 0 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

212 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MS2 Data Acquisition (1st peak triggered in DDA) MS/MS spectrum Precursor 851.95 m/z

georg_hamel_011713_12503_chymo_tubA_025dda #4329 RT: 39.20 AV: 1 NL: 7.09E6 T: FTMS + c NSI d Full ms2 [email protected] [111.00-1715.00] x5 279 100 90 peptide fragment ions peak 80

60 727 280 50 812

40 410 628 911 509 Relative Abundance 30 226 159 187 325 491 794 826 385 527 709 912 20 610 893 1011 1098 412 956 728 10 136 241 0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z

213 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Tandem Mass Spectrometry (MS/MS)

• Select Precursor Peptide • Isolate Precursor ions in Collision Cell • Fragment precursor ions with Collision Induced Dissociation (CID) • Measure m/z of Product ions

214 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Collision Induced Dissociation (CID or HCD)

Q1 q2 Detector Quadrupole Mass Filter Collision Cell

+LLLY + LLL N + + 2 N LLLYSSQICK 2 SSQICK+ Measure m/z values of +LLLYSSQICK+ QICK+ CK+ +LLLYS product ions N2

PRECURSOR ion 612.8 m/z (LLLYSSQICK) filtered in Q1

N2 collision gas

Collision Induced Dissociation Tandem Mass Spectrometry 1) Electric potential applied to collision cell 1st measurement (MS1) = Intact Peptide m/z 2) Ions accelerated to high kinetic energy 2nd measurement (MS2) = Peptide fragment ion m/z 3) Ions collide with neutral gas molecule values 4) Kinetic energy is converted to internal energy 5) Chemical bond breakage occurs

215 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Predictable Fragment Ions Types from Peptide Dissociation

matrixscience.com

peptide bonds

• Peptide Backbone has 3 Bond Types (peptide bond is the weakest of the 3 bonds) • Bond Breakage Yields Complimentary Ion Types, e.g., b- and y-type

216 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide Fragment Ions (or product ions)

Peptide MQIFVKTLTK • 604.8572 m/z precursor ion, monoisotopic • 1208.7077 Intact Peptide Mass, monoisotopic m/z b series y series m/z b9 1062.6 MQIFVKTLT K 147.1 y1 b8 961.6 MQIFVKTL TK 248.1 y2 b7 848.5 MQIFVKT LTK 361.3 y3 b6 747.4 MQIFVK TLTK 462.3 y4 b5 619.3 MQIFV KTLTK 590.4 y5 b4 520.3 MQIF VKTLTK 689.5 y6 b3 373.2 MQI FVKTLTK 836.5 y7 b2 260.1 MQ IFVKTLTK 949.6 y8 b1 132.0 M QIFVKTLTK 1077.7 y9

217 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide Tandem MS can provide Unambiguous Evidence for Location of Amino Acid Modifications

Rule: Experimental data must contain fragment ions that provide site localization evidence

218 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Tandem MS (or MS/MS) for Identification of Amino Acid Post-translational Modification (PTM) Site

MQIFVKTLTK MWmono = 1208.7077 MQIFVKTpLTK MWmono = 1288.6740 (phosphorylation +80 Da = HPO3) m/z b series y series m/z 1062.6 MQIFVKTLT K 147.1 961.6 MQIFVKTL TK 248.1 848.5 MQIFVKT LTK 361.3 747.4 MQIFVK TLTK 462.3 619.3 MQIFV KTLTK 590.4 520.3 MQIF VKTLTK 689.5 373.2 MQI FVKTLTK 836.5 260.1 MQ IFVKTLTK 949.6 132.0 M QIFVKTLTK 1077.7 Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed

219 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Predictable Mass Shifts for Phosphorylated Fragments MQIFVKTpLTK

m/z b series y series m/z +80 1062.6 MQIFVKTLT K 147.1 +80 961.6 MQIFVKTL TK 248.1 +80 848.5 MQIFVKT LTK 361.3 747.4 MQIFVK TLTK 462.3 +80 619.3 MQIFV KTLTK 590.4 +80 520.3 MQIF VKTLTK 689.5 +80 373.2 MQI FVKTLTK 836.5 +80 260.1 MQ IFVKTLTK 949.6 +80 132.0 M QIFVKTLTK 1077.7 +80 Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed

220 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Predictable Mass Shifts for Phosphorylated Fragments

MQIFVKTpLTK (+80 = HPO3); MWmono = 1288.67

m/z b series y series m/z 1142.6 MQIFVKTpLT K 147.1 1041.5 MQIFVKTpL TK 248.1 928.4 MQIFVKTp LTK 361.3 747.4 MQIFVK TpLTK 542.3 619.3 MQIFV KTpLTK 670.4 520.3 MQIF VKTpLTK 769.4 373.2 MQI FVKTpLTK 919.5 260.1 MQ IFVKTpLTK 1029.6 132.0 M QIFVKTpLTK 1157.6

Mass shifts will occur only in fragments containing the phos-Thr (bold), therefore location of MODIFICATION can be pinpointed

221 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

EXAMPLE: MS2 Spectrum and Confirmed Peptide Match

a=3.56742302613418490e-004, t0=4.31796646529983260e+001

2116

a2b2

199.1716 2000

1800 L LL Y S S Q I Ccam K Protein ID: Tyrosine-protein kinase JAK3

1600 (human) a1

y2 1400

86.0972

307.1400

1200 1000

800

y6 b3

136.0725

600

249.1534

722.3468 340.2637

y7 y8

147.1153

400 420.2225

885.3852 998.4837

y5 277.1471

200

y4 289.1373

635.3242

y8(2+) a4 a3-NH3(2+) a5

y7(2+) b4 y9

100 200 300 400 500 600 700 800 900 1000 1100 1200 m/ z, amu

222 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Intact Protein Electrospray Mass Spectrometry

223 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Intact Protein Electrospray - Mass Spectrum

+12 100 1031.0 +13 951.8 +11 1124.6 PROTEIN +8 1545.7 CHARGE +14 +10 884.0 1236.9 50 +9 SERIES 1374.2 +7

Relative Abundance Relative 1766.6 +15 825.0 +17 +16 727.5 772.4 0 2000 200 1000 m/z 12359 +/- 2 100

Mathematically 50

deconvoluted Abundance Relative Data  Protein MW

0 12500 15000 10000 mass

224 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Intact Protein ESI-MS Cytochrome C, 12361 Da

Charge = +8 NH3+ H+ H+ H+ H+ m 12361 + 12 H+ COOH = = 1031 H+ H+ z 12 +12 1031.0 m 12361 + 8 100 = = 1546 z 8 +8 1545.7

50 Relative Abundance Relative

0 200 1000 2000 m/z

225 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Intact Protein Electrospray-MS Infusion of Solubilized Relatively Pure Protein

• Relatively pure sample • 20 – 50 µM protein concentration • Detergent and salt free solution • Typical solvent: 50:50, acetonitrile:water, 0.1% formic acid • Difficult (but possible) to achieve high quality data • Non-covalent interactions are retained with ESI (not MALDI), usually with neutral/basic buffer system and a lot of trial and error

226 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Glycosylated Intact Protein ESI Mass Spec

1792 8000 1730 1858

6000 1672 Intermediate 1 1930 Protein

4000 1618 2007 Intensity, counts Intensity, 2000 1568 1753 1815 1882 2090

0 1600 1800 2000 2200 m/z

227 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Intermediate 1: Deconvoluted MS Data Using Bayesian Reconstruct Tool; Theoretical mass = 50140.01 Da; error=4 ppm 50140 .23 NAcNeu 1.0e5 | Gal Sialic acid | Fucose 8.0e4 GlcNAc

∆ 291 ∆ 656 ∆146 6.0e4

+K Intensity, cps Intensity, 4.0e4 50180

2.0e4 50229 50797 49849 50497 +K 50943 0 5.00e4 5.05e4 5.10e4 Mas s , amu

228 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

Wednesday AM Lecture 3: Specialized Applications in Mass Spectrometry

Bruce Witthuhn

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Quantification of small molecules, peptides and proteins

Survey (MS relative between samples) Targeted (MSMS) Absolute Metabolomics Proteomics • Focus - Selected Reaction Monitoring(SRM)/Multiple Reaction Monitoring (MRM) scan Mode as method of quantitation • External or internal standards

© 2015 Regents of the University of Minnesota. All rights reserved. 229 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Survey Scan (MS of intact molecule)and Identification (MSMS)

•Accurate mass •Retention time •Product Ions •Ultimately Relies on matching these to the known compound

© 2015 Regents of the University of Minnesota. All rights reserved. 230 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 © 2015 Regents of the University of Minnesota. All rights reserved. 231 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 © 2015 Regents of the University of Minnesota. All rights reserved. 232 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Target: Selective Reaction Monitoring

Relies on intact M/Z Relies on product ions produced under optimized conditions Relies on retention time

Capillary LC column

Q2 Q0 (Collision cell) Q3/LIT Skimmer EM Q1 Detector

Example 1: Quantification –MRM mode no internal standard Example 2: Quantification –MRM mode use of stable isotope labeled standard

© 2015 Regents of the University of Minnesota. All rights reserved. 235 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 © 2015 Regents of the University of Minnesota. All rights reserved. 236 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 FIGURE 1. The secondary structure of E. coli tRNALys(UUU) (a) and the chemical structure of t6A (b). THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 17, pp. 13666–13673, April 20, 2012 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

© 2015 Regents of the University of Minnesota. All rights reserved. 237 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Selected Reaction Monitoring in a Triple Quadrupole

Q1 (413) Q2 collision cell Q3(280.9)

LC column

Set on mass of Fragment precursor Transmit only precursor ion ion diagnostic (T6A) product ion

All fixed  Very Selective Scan

Bulk purification of tRNA Enzymatically hydrolysed to nucleotides Purified by running zip-tip c18 LC on C18 RP in presence of ammonium acetate MRM analysis T6A 413> 280.9 and 136 Adenosine 268>136 Ratio of T6A to Adenosine was determined

© 2015 Regents of the University of Minnesota. All rights reserved. 239 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 XIC of +MRM (4 pairs): 267.000/136.000 Da ID: adenosine_136 from Sample 22 (jixxx002_leixx039_11855_20120620_100000fm) of jix... Max. 130.0 cps.

1.3e4

1.0e4

5000.0 In te n sity, cp s

8.20 8.31 8.34 8.42 8.58 8.608.648.68 8.73 8.75 8.84 8.88 8.91 8.97 9.12 9.209.24 9.33 9.35 9.37 9.49 9.54 9.59 0.0 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 9.0 9.1 9.2 9.3 9.4 9.5 9.6 9.7 Time, min XIC of +MRM (4 pairs): 413.000/281.000 Da ID: T6A_281 from Sample 22 (jixxx002_leixx039_11855_20120620_100000fm) of jixxx002... Max. 1.3e4 cps.

8.82 1.3e4

1.0e4

5000.0 413>281 In te n sity, cp s

0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 268.000/136.000 Da ID: adenosine_136 from Sample 22 (jixxx002_leixx039_11855_20120620_100000fm) of ji... Max. 3090.0 cps.

8.73 3000

2000

1000

In te n sity,268>136 cp s

9.56 0 2 4 6 8 10 12 14 16 18 20 22 Time, min © 2015 Regents of the University of Minnesota. All rights reserved. 240 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 XIC of +MRM (4 pairs): 267.000/136.000 Da ID: adenosine_136 from Sample 17 (jixxx002_leixx039_11855_20120620_10000fm) of jixx... Max. 100.0 cps.

2810

2000

1000

6.98In7.077.23 te7.36 n7.46 s7.63 ity7.86 7.96 , 8.05c8.10 p8.24 s 8.45 8.61 8.85 8.90 9.18 9.22 9.459.54 9.62 9.84 10.04 10.19 10.32 10.48 0 7.0 7.2 7.4 7.6 7.8 8.0 8.2 8.4 8.6 8.8 9.0 9.2 9.4 9.6 9.8 10.0 10.2 10.4 10.6 Time, min XIC of +MRM (4 pairs): 413.000/136.000 Da ID: T6A_136 from Sample 17 (jixxx002_leixx039_11855_20120620_10000fm) of jixxx002... Max. 1060.0 cps.

8.81 1000 413/136 500

In te n s ity , c p s 9.739.88 10.62 11.40 13.66 14.37 16.2916.49 0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 268.000/136.000 Da ID: adenosine_136 from Sample 17 (jixxx002_leixx039_11855_20120620_10000fm) of jix... Max. 2810.0 cps.

8.71 2810

2000 268/136

1000

In te n s ity , c p s 8.92 9.78 16.24 16.81 0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 413.000/281.000 Da ID: T6A_281 from Sample 17 (jixxx002_leixx039_11855_20120620_10000fm) of jixxx002... Max. 1350.0 cps.

8.81 1350 1000 413/281

500

In1.39 te n s ity6.12 , c p7.89 s 9.069.58 11.06 13.55 14.4414.64 22.76 0 2 4 6 8 10 12 14 16 18 20 22 Time, min

© 2015 Regents of the University of Minnesota. All rights reserved. 241 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 © 2015 Regents of the University of Minnesota. All rights reserved. 242 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Wild-type XIC of +MRM (4 pairs): 267.000/136.000 Da ID: adenosine_136 from Sample 3 (jixxx002_leixx039_11855_20120620_W29) of jixxx002... Max. 1690.0 cps.

8.3e6

5.0e6 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 413.000/281.000 Da ID: T6A_281 from Sample 3 (jixxx002_leixx039_11855_20120620_W29) of jixxx002_leixx0... Max. 1.8e5 cps.

8.84 1.8e5 1.5e5 413/281 1.0e5 5.0e4 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 413.000/136.000 Da ID: T6A_136 from Sample 3 (jixxx002_leixx039_11855_20120620_W29) of jixxx002_leixx0... Max. 1.2e5 cps.

8.84 1.00e5

5.00e4 413/136 In te n s ity , c p s 0.00 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 268.000/136.000 Da ID: adenosine_136 from Sample 3 (jixxx002_leixx039_11855_20120620_W29) of jixxx002_... Max. 8.3e6 cps.

8.72 8.3e6 268/136 5.0e6 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min © 2015 Regents of the University of Minnesota. All rights reserved. 243 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Y - uninduced

XIC of +MRM (4 pairs): 267.000/136.000 Da ID: adenosine_136 from Sample 27 (jixxx002_leixx039_11855_20120620_Y) of jixxx002_l... Max. 1010.0 cps.

6.0e6

4.0e6

2.0e6 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 413.000/136.000 Da ID: T6A_136 from Sample 27 (jixxx002_leixx039_11855_20120620_Y) of jixxx002_leixx039... Max. 1.2e4 cps.

8.82 1.00e4 413/136

5000.00 In te n s ity , c p s 0.00 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 413.000/281.000 Da ID: T6A_281 from Sample 27 (jixxx002_leixx039_11855_20120620_Y) of jixxx002_leixx039... Max. 1.8e4 cps.

8.81 1.8e4 1.5e4 1.0e4 413/281 5000.0 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 268.000/136.000 Da ID: adenosine_136 from Sample 27 (jixxx002_leixx039_11855_20120620_Y) of jixxx002_le... Max. 6.5e6 cps.

8.71 6.0e6

4.0e6 268/136

2.0e6 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min

XIC of +MRM (4 pairs): 267.000/136.000 Da ID: adenosine_136 from Sample 31 (jixxx002_leixx039_11855_20120620_Yiptg) of jixxx00... Max. 870.0 cps.

6.0e6

4.0e6

2.0e6 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 413.000/281.000 Da ID: T6A_281 from Sample 31 (jixxx002_leixx039_11855_20120620_Yiptg) of jixxx002_leixx... Max. 1.0e5 cps.

8.84 1.00e5

5.00e4 413/281 In te n s ity , c p s 0.00 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 413.000/136.000 Da ID: T6A_136 from Sample 31 (jixxx002_leixx039_11855_20120620_Yiptg) of jixxx002_leixx... Max. 6.9e4 cps.

8.84 6.0e4 413/136 4.0e4

2.0e4 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min XIC of +MRM (4 pairs): 268.000/136.000 Da ID: adenosine_136 from Sample 31 (jixxx002_leixx039_11855_20120620_Yiptg) of jixxx00... Max. 6.6e6 cps.

8.73 6.0e6 268/136 4.0e6

2.0e6 In te n s ity , c p s 0.0 2 4 6 8 10 12 14 16 18 20 22 Time, min

© 2015 Regents of the University of Minnesota. All rights reserved. 245 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Determined that the T6A had increased when a target gene was induced with an IPTG inducible promoter.

Used ratio of an unlabeled internal standard housekeeping small molecule with similar characteristics as the target molecule.

Separated by LC

© 2015 Regents of the University of Minnesota. All rights reserved. 246 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 © 2015 Regents of the University of Minnesota. All rights reserved. 247 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Multiple Reaction Monitoring in QTRAP

Q1 Precursor mass Q2 collision cell Q3 product ions

75 44 Detection 58 Capillary LC 58 column 44 44 55 72 55

Transmit only CE optimized to only diagnostic precursor m/z produce diagnostic product ions of Selected to Q2 ions acrylamide

© 2015 Regents of the University of Minnesota. All rights reserved. 248 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Chemical & Engineering News Copyright © 2002 American Chemical Society

© 2015 Regents of the University of Minnesota. All rights reserved. 249 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Acrylamide Experimental design Extract acrylamide from french fries

Spike with stable isotope

Process samples with SPE column

LCMSMS on product ions 72>55 and 75>58

XIC of +MRM (4 pairs): 75.000/44.000 Da ID: acryl75/44 from Sample 126 (rosen006_mattm_11969_X636) of rosen006_mattm_11969... Max. 1250.0 cps.

7.7e4

6.0e4

4.0e4

2.0e4In te n sity, cp s

0.09 0.45 0.95 1.25 1.81 2.42 2.65 2.80 3.43 4.06 4.15 4.29 4.90 5.53 5.81 6.17 6.54 6.81 6.91 7.46 7.84 8.52 8.75 8.92 9.65 9.82 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time, min XIC of +MRM (4 pairs): 75.000/58.000 Da ID: acryl75/58 from Sample 126 (rosen006_mattm_11969_X636) of rosen006_mattm_11969_... Max. 7.7e4 cps.

2.66 7.7e4 75/58 6.0e4

4.0e4

2.0e4In te n sity, cp s 2.42

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time, min XIC of +MRM (4 pairs): 72.000/55.000 Da ID: acryl72/55 from Sample 126 (rosen006_mattm_11969_X636) of rosen006_mattm_11969_... Max. 7.4e4 cps.

2.66 7.4e4

6.0e4

4.0e4 72/55

2.0e4In te n sity, cp2.42 s 0.28 2.06 2.80 0.43 0.90 1.48 1.59 3.19 3.83 4.104.35 4.81 5.35 5.46 5.72 5.81 6.14 6.43 6.78 7.097.33 7.59 8.31 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time, min

Sample Name Conc Area Area Ratio Calc Conc Accuracy 575 N/A 438710.7661 0.696485206 668.2531505 N/A 575dup N/A 405065.4651 0.597388917 573.8170576 N/A 583 N/A 1258691.35 1.86163436 1778.608893 N/A 583dup N/A 1181993.718 1.65007301 1576.996628 N/A 619 N/A 360802.5102 0.482302645 464.142942 N/A 619dup N/A 492556.4521 0.705858204 677.1853648 N/A standard 100pg 100 171811.6321 0.099074211 98.93657198 98.93657198 standard 200pg 200 537783.4157 0.207571201 202.3312791 101.1656395 standard 400pg 400 907843.3899 0.397525022 383.352152 95.838038 standard 600pg 600 1191880.348 0.651920405 625.7840957 104.2973493 standard 800pg 800 1782703.46 0.82604448 791.719647 98.96495588 standard 1000pg 1000 3218396.886 1.033375724 989.3007344 98.93007344 standard 100pg 100 199147.6783 0.097779106 97.70237165 97.70237165 standard 200pg 200 577656.6349 0.206046147 200.8779445 100.4389722 standard 400pg 400 1029899.267 0.40132209 386.9706552 96.7426638 standard 600pg 600 1318852.46 0.658099536 631.6726406 105.2787734 standard 800pg 800 1980643.766 0.83004524 795.5322633 99.44153292 standard 1000pg 1000 3399541.906 1.037169871 992.9164546 99.29164546

© 2015 Regents of the University of Minnesota. All rights reserved. 253 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MS/MS Provides Information for MRM Transitions

+EPI (724.37) Charge (+2) CE (44.2186) FT (25): Exp 3, 49.843 min from Sample 1 (1 ug) of porte001_porte001_FAP50_MIDASrun1_... Max. 2.0e6 cps.

425.3 ProteinPilot interpretation of MS/MS data 2.0e6 1.9e6 MS/MS data from 1.8e6

1.7e6 captured peptide

1.6e6 510.4

1.5e6

1.4e6 234.4 1.3e6 811.4 1.2e6120.1

1.1e6 910.6

1.0e6

Intensity,9.0e5 cps 130.2

8.0e5 696.5 1023.7 7.0e5 217.3 175.2 6.0e5 407.4 1186.8 5.0e5 249.4 4.0e5 366.4 625.3 538.4 3.0e5 262.2 394.4 724.4 2.0e5 158.0 321.4 893.5 1109.9 226.2 493.4 584.4 608.2 845.8 920.6 336.4 634.6 996.8 1.0e5 141.0 342.0 828.5 1154.6

0.0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 Precursor m/z 724.3 m/z, Da

XIC of +MRM (15 pairs): 450.2/786.4 Da ID: LCVLHEK from Sample 1 (100 fmol) of nelse002_ander234_BSARM_1208091.wiff (Nano... Max. 1490.0 cps.

1.29e6 1.25e6 1.20e6 1.15e6 1.10e6 1.05e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5

Intensity,5.50e5 cps 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4 35.84 0.00 34.5 35.0 35.5 36.0 36.5 37.0 37.5 38.0 38.5 39.0 39.5 40.0 40.5 41.0 41.5 42.0 42.5 43.0 43.5 44.0 44.5 45.0 45.5 1315 1334 1353 1372 1391 1410 1429 1448 1467 1486 1505 1524 1543 1562 1581 1600 1619 1638 1657 1676 1695 1714 1733 Time, min

Q1 Dwell Q1 Q3 Dwell Mass Q3 Mass Time ID CE Mass Mass Time ID CE 450.2 786.4 100 LCVLHEK 31.7 554.8 600.3 100 EACFAVEGPK 29.8 461.7 Y6/722.4 100 AEFVEVTK 31.4 570.7 818.4 100 LVNELTEFAK 27.5 464.2 Y5/651.3 100 YLYEIAR 41.5 571.9 886.6 100 KQTALVELLK 28.1 474.2 552.3 100 SLHTLFGDELCK 40.7 582.3 951.5 100 LVNELTEFAK 28.8 480.6 587.4 100 RHPEYAVSVLLR 33.4 628 948.5 100 RPCFSALTPDETYVPK 28.7 487.7 Y6/746.4 100 DLGEEHFK 49 653.4 1055.6 100 HLVDEPQNLIK 29 547.3 589.3 100 KVPQVSTPTLVEVSR 32.1 722.8 1168.5 100 YICDNQDTISSK 33.6

© 2015 Regents of the University of Minnesota. All rights reserved. 255 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Phosphorylated Protein Detection by Mass Spectrometry

Challenges • Low abundance of modified protein (relative to unmodified or other proteins) • MS signal suppression • Ionization efficiency • Data analysis inefficiency (software limitations) • Phosphate group is labile under mass spectrometric conditions Methods for success • Enrichment • Variation of proteolytic enzymes • Both ESI and MALDI MS • Multiple software packages and manual interpretation

© 2015 Regents of the University of Minnesota. All rights reserved. 256 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 ENRICHMENT for PHOSPHOPEPTIDES May be Crucial to Success

Dual enrichment method 1. SCX separation: at pH 2.7, phosphopeptides elute early 2. Immobilized Metal Affinity Chromatography (IMAC)

IDENTIFICATION of 1000’s of phosphopeptides/proteins Villén J & Gygi SP 2008, Nature Protocols 3(10), 1630 © 2015 Regents of the University of Minnesota. All rights reserved. 257 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 a. i . Crude Peptides

25000

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0 500 1000 1500 2000 2500 3000 m/ z © 2015 Regents of the University of Minnesota. All rights reserved. / I =/ aug05/ baw080505phosphomi x_crude/ 1Ref / pdat a/ 1 Admi ni st rat or Wed Aug 24 16:258 30: 42 2005 Center for Mass Spectrometry and Proteomics | PhoneSudha | Marimanikkuppam(612)625-2280 (University| (612)625 of- 2279Minnesota) a. i . Peptides Zip Tipped C18

5500

5000

4500

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3500 Phosphopeptide 3000

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1300 1800 2300 m/ z © 2015 Regents of the University of Minnesota. All rights reserved. / I =/ aug05/ baw080505phosphomi x_zt / 1Ref / pdat a/ 1 Admi ni st rat or Wed Aug 24 16: 33:259 03 2005 Center for Mass Spectrometry and Proteomics | PhoneSudha | Marimanikkuppam(612)625-2280 (University| (612)625 of- 2279Minnesota) a. i . Peptides Titania Tip

3500 Phosphopeptide

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1400 1900 2400 m/ z © 2015 Regents of the University of Minnesota. All rights reserved. / I =/ aug05/ baw080505phosphomi x_t i t ani a/ 1Ref / pdat a/ 1 Admi ni st rat or Wed Aug 24 16:260 34: 51 2005 Center for Mass Spectrometry and Proteomics | PhoneSudha | Marimanikkuppam(612)625-2280 (University| (612)625 of- 2279Minnesota)

Wednesday Workshop F. Manual interpretation of MS/MS spectra

Candace Guerrero

• The information contained in an MS spectrum (m/z, isotope spacing and therefore “z”) is not enough to tell us the amino acid sequence • Luckily, peptides fragment in a predictable way and a spectrum of these fragments can help us figure out the sequence

• 100,000’s of molecules of a particular peptide will be isolated and fragmented • Each of these molecules will create a b / y ion pair in an MS/MS spectrum • The spacing (mass difference) between peaks in an MS/MS spectrum is characteristic of the amino acid residue(s) differing between the two sequences

Immonium Ion

• Glycyl-glycine (GG)

minimal fragmentation more fragmentation 133.06 76.04 100 100 + 90 [Gly-gly + H] 90 [Gly + H]+ 80 80 70 70 60 60 50 76.04 50 40 Δ m/z = 40 30 + 30

Relative Relative intensity [Gly + H] 57.02 Relative intensity + 20 20 [Gly-gly + H] 10 10 133.06 0 0 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 140 m/z m/z

The spacing of these peaks (57.021 m/z) tells us that the heavier ion has an extra glycine residue compared to the lighter ion

Look for the b2/a2 pair, Δ m/z = 28

Neutral loss of H2O (-18) and NH3 (-17) are relatively common © 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 The Nine-Step Strategy for Interpretation (from Reference 1) 1. Inspect the low-mass region for immonium ions. The first step in the interpretation is to inspect the low-mass region of the spectrum, noting the presence of any immonium ions and the amino acid composition that they indicate.

2. Inspect the low-mass region for the b2-ion. In the second step of the interpretation, the low-mass region of the spectrum is inspected to identify the b2-ion, generally recognizable by the b2-ion / a2-ion pair separated by 28 Da. By using Table 4.2, the possible two-amino acid-combinations indicated by the b2-ion are noted. The m/z of the b2-ion is then used to calculate the m/z of the corresponding yn-2-ion, and the high-mass region of the product ion spectrum is inspected to identify this ion.

3. Inspect the low-mass region for the y1-ion. The third step of the interpretation is to assign the C-terminal amino acid. The low-mass region of the spectrum is inspected to identify the y1-ion at either m/z 147, for C-terminal lysine peptides, or m/z 175, for C-terminal arginine peptides. The m/z of the y1 -ion is then used to calculate the m/z of the bn-1-ion, and the high-mass region of the product ion spectrum is inspected to identify that ion, if present.

4. Inspect the high-mass region to identify the yn-1-ion. The fourth step of the interpretation is to attempt to assign the N- terminal amino acids from combinations indicated by the b2-ion. The high-mass region of the spectrum is scrutinized to identify the yn-1-ion, if present. The list of possible amino acid combinations derived from the b2-ion limits the possible residue masses to consider. If an ion is identified the m/z of that ion is used to calculate the residue masses of the first two amino acids and to assign those peptides. 5. Extend the y-ion series toward lower m/z. Working with the residue masses listed in Table 4.1, begin to extend the y-ion series backwards (toward lower m/z) from the yn-2-ion. As a y-ion is identified calculate the m/z of the corresponding b-ion and identify that ion in the spectrum. Work towards extending the y-ion series from the yn-2 ion to the y1-ion 6. Extend the b-ion series toward higher m/z. If progress extending the y-ion series falters, use the residue masses listed in Table 4.1 to extend the b-ion series from the last identified b-ion. As any b-ions are identified, use the m/z of that ion to calculate the m/z of the corresponding y-ion and identify that ion in the spectrum. 7. Calculate the mass of the peptide. When the interpretation of the spectrum is complete, calculate the mass of the proposed peptide sequence and check its agreement with the measured mass. 8. Reconcile the amino acid content with spectrum data. Check that the amino acid content agrees with the immonium ions observed. Also consider the charge state of the peptide in terms of the presence of histidine, and internal lysine or arginine residues. 9. Attempt to identify all ions in the spectrum. Work to identify the other ions in the spectrum based on the proposed peptide sequence and pay particular attention to the ions from the loss of© H 20152O, NH Regents3, and HSOCHof the University3; any doubly of Minnesota. charged ions; All rightsand any reserved. ions due to internal cleavages. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 LeeAnn just showed you: Peptide MS/MS Spectrum Precursor [M + 2H]+2 698.86 m/z

Charge state information is calculated from full scanhegem007_fanx0092_120812_12508_seed_soluble #3755 RT: 36.50 AV: 1 NL: 1.11E5 T: FTMS + c NSI Full ms [360.00-1800.00] 698.86 z=2 100 90 Full scan 80 699.36 hegem007_fanx0092_120812_12508_seed_soluble #3757 RT: 36.52 AV: 1 NL: 3.74E3 698.86 m/z z=2 70 T: FTMS + c NSI d Full ms2 [email protected] [111.00-1410.00] precursor 708.37 60 100 50 isotope

40 Relative Relative Abundance series: z = 2 699.86 90 30 z=2 20

894.47 10

175.12 0 80 698.0 698.2 698.4 698.6 698.8 699.0 699.2 699.4 699.6 699.8 700.0 700.2 700.4 700.6 m/z 508.29 70

229.12 60

807.44 50 263.10 300.16 995.52 40

RelativeAbundance 392.19 637.33 1082.55 30

1283.63 20 337.11 1196.59 10 1046.53 463.16 550.16 951.49

0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 m/z© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MS/MS Spectrum [M + 2H]+2 698.86 m/z 400 – 1400 m/z Calculate a Partial Amino Acid Sequence from the consecutive y-ion series

Start: hegem007_fanx0092_120812_12508_seed_soluble #3757 RT: 36.52 AV: 1 NL: 3.74E3 T: FTMS + c NSI d Full ms2 [email protected] [111.00-1410.00] +1 708.37 [M + H] – 1283.63 = _____ (residue mass) 100 1283.63 – 1196.59 = ______(residue mass) 90 894.47 80

508.29 70

807.44 50 995.52 40 409.22 RelativeAbundance 637.33 1082.55 30

1283.63 20 1196.59 10

0 400 500 600 700 800 900 1000 1100 1200 1300 m/z

© 2015 Regents of the University of Minnesota. All rights reserved. FurtherCenter forreference: Mass Spectrometry http://www.ionsource.com/tutorial/DeNovo/b_and_y.htm and Proteomics | Phone | (612)625-2280 | (612)625-2279 MS/MS Spectrum [M + 2H]+2 698.86 m/z Low m/z range

hegem007_fanx0092_120812_12508_seed_s... 12/8/2012 11:43:13 PM

hegem007_fanx0092_120812_12508_seed_soluble #3757 RT: 36.52 AV: 1 NL: 3.04E3 T: FTMS + c NSI d Full ms2 [email protected] [111.00-1410.00] 120.08 100 175.12

80 136.08 202.08 70

263.10 60 300.16 50 235.11 245.12 40

RelativeAbundance 158.09 30 129.10 271.10 289.11 20 183.11 219.08 240.10 207.11 229.12 10

0 120 140 160 180 200 220 240 260 280 300 m/z

bhering_dejon039_082912_12156_preiTQ_0989 #5252 RT: 38.96 AV: 1 NL: 9.80E4 T: FTMS + c NSI d Full ms2 [email protected] [111.00-1655.00] 288.20 100

75 258.11 70

RelativeAbundance 40 213.09 35 175.12 30 185.09 169.13 25 1074.52 20 159.09 312.16 502.30 817.41 1003.48 1203.57 15 440.22 1314.60 444.27 1331.62 10 617.33 329.15 746.37 403.23 569.26 1185.56 5 640.29 1430.70 842.44 986.47 1296.58 1395.65 1527.78 0 200 300 400 500 600 700 800 ©900 2015 1000Regents1100 of the University1200 1300 of Minnesota.1400 1500 All rights1600 reserved. m/z Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Example #3

tgriffin_dejon039_073013_13290_6c #4453 RT: 30.58 AV: 1 NL: 3.02E4 T: FTMS + c NSI d Full ms2 [email protected] [111.00-1505.00] 245.08 100 217.08 95

60 175.12

RelativeAbundance 40

35 635.28 506.24 30 377.20 763.38 25 894.42 1008.46 20 1119.49 518.23 15 991.43 259.09 1136.52 389.18 10 360.17 617.27 746.35 773.36 468.19 1102.47 5 312.16 596.25 877.39 974.41 645.27 835.32 1069.74 1249.60 0 200 300 400 500 600 700 800© 2015900 Regents1000 of the University1100 1200 of Minnesota.1300 All1400 rights1500 reserved. m/z Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 References

1. Protein Sequencing and Identification Using Tandem Mass Spectrometry: http://onlinelibrary.wiley.com/book/10.1002/0471721980 and especially chapter 4, Collisionally induced dissociation of protonated peptide ions and the interpretation of product ion spectra: http://onlinelibrary.wiley.com/doi/10.1002/0471721980.ch4/pdf 2. http://www.ionsource.com/tutorial/DeNovo/b_and_y.htm

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Thursday AM Lecture 1: Protein Sequence Databases …and your Mass Spectrometry-based Proteomics Experiment

LeeAnn Higgins

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Outline Terminology • Protein Database (DB) • FASTA • Origin • Database repository • Sources • Format • NCBI database • Size • UniProtKB • Composition • Swiss Prot • Selecting a database for mass spec search • Ref Seq (reference sequence) • Effect of DB on mass spec search results • Homology • Post MS analysis: protein • Contaminants DB annotation, ontology, alignment • Ontology

© 2015 Regents of the University of Minnesota. All rights reserved. 276 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 FASTA Protein Sequence • Name and Origin • FASTA (pronounced ‘fast-aye’) • ORIGIN: for sequence similarity alignment tool (1985) • REF: DJ Lipman, WR Pearson (1985) PMID: 2983426 "The algorithm has been implemented in a computer program designed to search protein databases very rapidly. For example, comparison of a 200-amino-acid sequence to the 500,000 residues in the National Biomedical Research Foundation library would take less than 2 minutes on a minicomputer, and less than 10 minutes on a microcomputer (IBM PC)." • Stands for “fast all” – the file format worked with ‘all’ alphabets (amino acid and nucleotide)

• Structure: TEXT file • Line 1: description line with sequence identifier • Line 2: single amino acid letter protein sequence 80 characters wide • Allowed characters: • AMINO ACID ONE-LETTER CODE • X • * • - • Custom one-letter amino acid codes

© 2015 Regents of the University of Minnesota. All rights reserved. 278 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Line 1: description line with sequence identifier FASTA Format Header Line Sequence Identifiers

© 2015 Regents of the University of Minnesota. All rights reserved. 279 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Line 2 FASTA Protein Sequence from NCBI- example

Line 1

Line 2

NOTE: In Sept 2016, gi numbers were replaced with accession.version identifiers

© 2015 Regents of the University of Minnesota. All rights reserved. 280 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Selecting a Protein Sequence Database • Public repositories, such as • NCBI • UniProtKB • Swiss Prot: manually annotated and reviewed • TrEMBL: Automatically annotated and not reviewed • Custom (from customer) • NOTE: format is important! • Represent species (1 or more) from which protein sample originated • Example: Mouse protein expressed in E. coli • Ideal size range ~ 2000 to < 1 million entries

© 2015 Regents of the University of Minnesota. All rights reserved. 281 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Selecting a Protein Database: UniProtKB repository

© 2015 Regents of the University of Minnesota. All rights reserved. 282 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Selecting a Protein Database: NCBI Ref Seq repository

© 2015 Regents of the University of Minnesota. All rights reserved. 283 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Choose Your Taxonomy or Taxonomies NOTES: • If recombinant protein expressed in host cell, include host proteins & expressed protein(s) • If protein database for your species has <2000 proteins, merge with another protein database (yeast) for statistical reasons • Protein sequence headers must be parsed correctly

(19996)

© 2015 Regents of the University of Minnesota. All rights reserved. 286 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Protein Database repository content for Thirteen-lined Ground Squirrel

Database Source Number of Proteins Swiss-Prot* reviewed 20 TrEMBL* unreviewed 20,076 UniProt Reference Proteome 19,966 NCBI (‘non-redundant’) 30,130 NCBI Reference Sequence 29,842

* From UniProt

© 2015 Regents of the University of Minnesota. All rights reserved. 287 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Protein Database Characteristics …related to your mass spectrometry experiment

© 2015 Regents of the University of Minnesota. All rights reserved. 288 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 SPLICES FORM variants Sequence alignments: Protein Cytochrome P450 2D6

Natural variants)

SNP’s (single nucleotide polymporphisms)

https://hive.biochemistry.gwu.edu

Peptide example Peptide Mass * Peptide Sequence 1 – native 1730.8443 SELEEQLTPVAEETR 1 – variant (Q -> K) 1730.8806 SELEEKLTPVAEETR 1 – variant (Q -> K) 734.3566 SELEEK 1 – variant (Q -> K) 1015.5418 LTPVAEETR

2 – native 830.4366 EQVAEVR 2 – variant (V -> E) 860.4108 EQEAEVR

* Monoisotopic [M + H]+1

© 2015 Regents of the University of Minnesota. All rights reserved. 293 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Proteomics Search Program Meets Protein Sequence Database • Protein sequence file is downloaded to local computer • Merge with common lab contaminants (keratins and more) database • http://www.thegpm.org/crap/ • Protein database is imported or indexed in the proteomics search program (sequence format is critical) • REVERSED sequences are generated for False Discovery Rate (FDR) calculations • Protein sequences are digested with enzymes in silico

• Database search algorithm matches spectrum > peptide > protein • RESULTS: List of protein identifications with accession numbers • POST Database search options (outside CMSP): 1. Protein annotation 2. Sequence alignment 3. Obtain related Gene Ontology information

© 2015 Regents of the University of Minnesota. All rights reserved. 295 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 POST Database search options What you can do with your protein list.

© 2015 Regents of the University of Minnesota. All rights reserved. 297 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2) Sequence alignment with UniProt alignment tool

© 2015 Regents of the University of Minnesota. All rights reserved. 298 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2) Sequence alignment with UniProt alignment tool: numerous amino acid labeling options

* (asterisk) indicates positions which have a single, fully conserved residue. : (colon) indicates conservation between groups of strongly similar properties - scoring > 0.5 in the Gonnet PAM 250 matrix. . (period) indicates conservation between groups of weakly similar properties - scoring =< 0.5 in the Gonnet PAM 250 matrix.

© 2015 Regents of the University of Minnesota. All rights reserved. 300 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3) Link Gene Ontology information to Proteins • Define: “The Gene Ontology (GO) project is a collaborative effort to address the need for consistent descriptions of gene products across databases.” • Ontologies/Vocabularies • molecular function: molecular activities of gene products • cellular component: where gene products are active • biological process: pathways and larger processes made up of the activities of multiple gene products

(http://geneontology.org/page/documentation)

© 2015 Regents of the University of Minnesota. All rights reserved. 301 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Molecular Function Pie Chart for a List of 96 Protein Identifiers (gi numbers) submitted to PANTHER (http://www.pantherdb.org/)

Protein list from Supplemental data REF: Thu TM et al (2016) Cell Reports, 15(6):1254-65; PMID: 27134171

© 2015 Regents of the University of Minnesota. All rights reserved. 302 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Protein Class Pie Chart for a List of 96 Protein Identifiers (gi numbers) submitted to PANTHER (http://www.pantherdb.org/)

Protein list from Supplemental data REF: Thu TM et al (2016) Cell Reports, 15(6):1254-65; PMID: 27134171

© 2015 Regents of the University of Minnesota. All rights reserved. 303 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Biological Process Pie Chart for a List of 96 Protein Identifiers (gi numbers) submitted to PANTHER (http://www.pantherdb.org/)

Protein list from Supplemental data REF: Thu TM et al (2016) Cell Reports, 15(6):1254-65; PMID: 27134171

• http://geneontology.org/ • http://string-db.org/ • http://www.pantherdb.org/ • http://www.ingenuity.com/products/ipa (licensed at UM via MSI)

ALSO: Match mass spec data to your RNA Seq data with: • https://galaxyp.msi.umn.edu/

Thursday AM Lecture 2: Protein Identification by Mass Spectrometry

LeeAnn Higgins

© 2015 Regents of the University of Minnesota. All rights reserved. Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Outline Terminology • Database search types • Mass tolerance • Peptide Mass Fingerprint • MS/MS search (PMF) • FASTA • Precursor mass-based • Sequence tag • Tandem MS • Precursor mass • Results comparison across programs • Product ion • Manual inspection of • Theoretical mass results • Experimental mass • Sequence tag • de novo sequencing

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Three Strategies for Protein Inference from Peptide MS or MS/MS Data

Peptide Mass Peptide MS/MS DATA INPUT: List (MS1) (MS2)

Peptide Mass Precursor Sequence- Search TYPE: Fingerprint Mass-based based (PMF) (historical) (de novo or 2 mass tag) 1 3

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide Mass Fingerprint (PMF) 1 • Historical: after 1D or 2D in-gel Digestion of single bands or spots • Current applications: limited; decreasing utility

Component Description Input (Data) Peptide Mass List • Mass type (Mr or MH+) • Average or Monoisotopic Target Protein FASTA Sequence Database Search Parameters Proteolytic enzyme # Missed enzyme cleave sites Amino acid modifications Mass tolerance

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 Peptide Mass Fingerprint: INPUT GLSDGEWQQVLNVWGK VEADIAGHGQEVLIR A) In gel Trypsin LFTGHPETLEK UNKNOWN PROTEIN (amino acid sequence): TEAEMK GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTG digestion ASEDLK HPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLT HGTVVLTALGGILK ALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLE GHHEAELKPLAQSHATK FISDAIIHVLHSHPGNFGADAQGAMTKALELFRND YLEFISDAIIHVLHSHPGNFGADAQGAMTK IAAKYKELGFQG ALELFR NDIAAK ELGFQG B) Acquire MALDI-TOF Mass Spectrum of peptide mixture

C) Experimental Peptide Mass (or m/z) List

m/z Peptide 631.71 NDIAAK 650.71 ELGFQG 662.72 ASEDLK 708.81 TEAEMK 748.90 ALELFR 1272.44 LFTGHPETLEK 1379.69 HGTVVLTALGGILK 1607.81 VEADIAGHGQEVLIR Absolute Intensity Absolute 1817.01 GLSDGEWQQVLNVWGK 1855.06 GHHEAELKPLAQSHATK m/z 3241.65 YLEFISDAIIHVLHSHPGNFGADAQGAMTK

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 PMF TARGET: Protein FASTA Sequence Database “ >sp|P31946|1433B_HUMAN 14-3-3 protein beta/alpha OS=Homo sapiens UniProt (example) GN=YWHAB PE=1 SV=3 The Universal Protein Resource (UniProt) is a comprehensive resource MTMDKSELVQKAKLAEQAERYDDMAAAMKAVTEQGHELSNEERNLLSVAYKNVVGARRSS WRVISSIEQKTERNEKKQQMGKEYREKIEAELQDICNDVLELLDKYLIPNATQPESKVFY for protein sequence and annotation data. UniProt is a collaboration LKMKGDYFRYLSEVASGDNKQTTVSNSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFY between the European Bioinformatics Institute (EBI), the Swiss Institute YEILNSPEKACSLAKTAFDEAIAELDTLNEESYKDSTLIMQLLRDNLTLWTSENQGDEGD AGEGEN of Bioinformatics (SIB) and the Protein Information Resource (PIR). >sp|P31946-2|1433B_HUMAN Isoform Short of 14-3-3 protein Across the three institutes close to 150 people are involved through beta/alpha OS=Homo sapiens GN=YWHAB MDKSELVQKAKLAEQAERYDDMAAAMKAVTEQGHELSNEERNLLSVAYKNVVGARRSSWR different tasks such as database curation, software development and VISSIEQKTERNEKKQQMGKEYREKIEAELQDICNDVLELLDKYLIPNATQPESKVFYLK support.” http://www.uniprot.org/help/about MKGDYFRYLSEVASGDNKQTTVSNSQQAYQEAFEISKKEMQPTHPIRLGLALNFSVFYYE ILNSPEKACSLAKTAFDEAIAELDTLNEESYKDSTLIMQLLRDNLTLWTSENQGDEGDAG EGEN Example: >sp|P62258|1433E_HUMAN 14-3-3 protein epsilon OS=Homo sapiens GN=YWHAE PE=1 SV=1 20,292 entries for taxonomy: "Homo sapiens (Human) [9606]“ MDDREDLVYQAKLAEQAERYDEMVESMKKVAGMDVELTVEERNLLSVAYKNVIGARRASW AND reviewed:yes RIISSIEQKEENKGGEDKLKMIREYRQMVETELKLICCDILDVLDKHLIPAANTGESKVF YYKMKGDYHRYLAEFATGNDRKEAAENSLVAYKAASDIAMTELPPTHPIRLGLALNFSVF YYEILNSPDRACRLAKAAFDDAIAELDTLSEESYKDSTLIMQLLRDNLTLWTSDMQGDGE EQNKEALQDVEDENQ >sp|Q04917|1433F_HUMAN 14-3-3 protein eta OS=Homo sapiens GN=YWHAH PE=1 SV=4 MGDREQLLQRARLAEQAERYDDMASAMKAVTELNEPLSNEDRNLLSVAYKNVVGARRSSW RVISSIEQKTMADGNEKKLEKVKAYREKIEKELETVCNDVLSLLDKFLIKNCNDFQYESK VFYLKMKGDYYRYLAEVASGEKKNSVVEASEAAYKEAFEISKEQMQPTHPIRLGLALNFS VFYYEIQNAPEQACLLAKQAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDE EAGEGN >sp|P61981|1433G_HUMAN 14-3-3 protein gamma OS=Homo sapiens GN=YWHAG PE=1 SV=2 MVDREQLVQKARLAEQAERYDDMAAAMKNVTELNEPLSNEERNLLSVAYKNVVGARRSSW RVISSIEQKTSADGNEKKIEMVRAYREKIEKELEAVCQDVLSLLDNYLIKNCSETQYESK VFYLKMKGDYYRYLAEVATGEKRATVVESSEKAYSEAHEISKEHMQPTHPIRLGLALNYS VFYYEIQNAPEQACHLAKTAFDDAIAELDTLNEDSYKDSTLIMQLLRDNLTLWTSDQQDD DGGEGNN >sp|P31947|1433S_HUMAN 14-3-3 protein sigma OS=Homo sapiens GN=SFN PE=1 SV=1 MERASLIQKAKLAEQAERYEDMAAFMKGAVEKGEELSCEERNLLSVAYKNVVGGQRAAWR VLSSIEQKSNEEGSEEKGPEVREYREKVETELQGVCDTVLGLLDSHLIKEAGDAESRVFY LKMKGDYYRYLAEVATGDDKKRIIDSARSAYQEAMDISKKEMPPTNPIRLGLALNFSVFH YEIANSPEEAISLAKTTFDEAMADLHTLSEDSYKDSTLIMQLLRDNLTLWTADNAGEEGG EAPQEPQS etc… Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 PMF Search Parameters Review: • Trypsin Proteolytic Enzyme and 1 • 0 Missed Cleave Site (#MC)

modifications

Example: Theoretical Trypsin Digest Peptide Mass List (truncated) for a Single Protein: Trypsin = Enzyme Missed Cleave Sites = 0

Center for Mass Spectrometry and Proteomics | Phone | (612)625http://www.expasy.org/tools/peptide-2280 | (612)625-2279 -mass.html PMF Search Parameters: • Trypsin Proteolytic Enzyme and 1 • 1 Missed Cleave Site • MSO = Methionine Oxidation (amino acid modification)

artif modifications modifications

Center for Mass Spectrometry and Proteomics | Phone | (612)625http://www.expasy.org/tools/peptide-2280 | (612)625-2279 -mass.html PMF Parameter: 1 Peptide Mass Deviation (+/- m/z)

Range: 631.339 – 631.351 3 Mouse proteins with High resolution measurement Trypsin peptide in this 631.345 +/- 0.006 range

197 Mouse proteins Low resolution measurement Range: 630 – 632 with Trypsin peptide in 631 +/- 1 this range

630 631 632 633 m/z

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 Mass Measurement Error Calculation Fractional Error Error Expression Expression Multiply by: ppm ∆ / Theoretical value 1,000,000 % ∆ / Theoretical value 100 where Δ = Experimental (or observed) – Theoretical m/z value Example: Experimental/observed value (i.e., the data acquired by the mass spectrometer) = 1480.107 m/z Theoretical value (calculated from periodic table, after the peak is identified) = 1480.028 m/z Delta (Δ) = 0.079

Error Expression Error Equation Error ppm (0.079/1480.028)*1,000,000 53 ppm % (0.079/1480.028)*100 0.0053%

A USEFUL REFERENCE: Brenton AG, Godfrey AR. Accurate mass measurement: terminology and treatment of data. J Am Soc Mass Spectrom. 2010 Nov;21(11):1821-35.

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 MASCOT Peptide Mass Fingerprint Search http://www.matrixscience.com/

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 MASCOT PMF Search Parameter Page

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 PMF Search m/z Peptide 631.71 NDIAAK 650.71 ELGFQG 662.72 ASEDLK 708.81 TEAEMK QUERY (Experimental Data) 748.90 ALELFR 1272.44 LFTGHPETLEK 1379.69 HGTVVLTALGGILK 1607.81 VEADIAGHGQEVLIR 1817.01 GLSDGEWQQVLNVWGK 1855.06 GHHEAELKPLAQSHATK 3241.65 YLEFISDAIIHVLHSHPGNFGADAQGAMTK

Compare QUERY to THEORETICAL Search PEPTIDE MASS LIST for each protein in the database Parameters: • Enzyme • Missed cleave site • Amino acid mods • Mass tolerance Score Probability-based MOWSE Score (often, the protein with highest number of peptide matches has the highest score)

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 1 PMF Search: Results Interpretation

• Is the protein ID experimentally rational? • Does the MW of protein in search results match MW determined by SDS-PAGE? • Does the pI of protein in search results match pI determined by 2D-PAGE?

Perform MS/MS if no protein match

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Three Strategies for Protein Inference from Peptide MS or MS/MS Data

Peptide Mass Peptide MS/MS DATA INPUT: List (MS1) (MS2)

Peptide Mass Precursor Sequence- Search TYPE: Fingerprint Mass-based based (PMF) (historical) (de novo or 2 mass tag) 1 3

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2 Tandem MS: Precursor Mass-based Search Component Description Input (Data) Precursor Mass & Product Ion Masses • Charge state • Average or Monoisotopic Target Protein FASTA Sequence Database Search Parameters Proteolytic enzyme # Missed enzyme cleave sites Amino acid modifications Mass tolerances: • Peptide/Precursor Mass • Product Ions Masses

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2 Tandem MS: Precursor Mass-based Search

1. Each MS/MS spectra has 2 data-rich Components

2. Database search for each MS/MS spectra has 2 STEPS

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Tandem MS: Precursor Mass-based Search 2 Calculate Precursor Mass from Precursor m/z Signal • Precursor m/z (monoisotopic) = 894.0941 • Precursor Charge State = 3 • Precursor Mass (Da) = 2679.2559

Precursor m/z Signal (intact peptide signal)

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2 Database search for Peptides

• Target peptide mass = precursor mass example = 2679.2559 • Target peptide mass range = precursor mass + / - precursor mass tolerance (user-specified) example = 50 ppm mass tolerance Example = 2679.2559 +/- 0.1340 Example = 2679.1219 – 2679.3899

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2 Peptide Candidate Matches for 2679.2559 precursor mass

Mr(expt) Mr(calc) ppm Peptide Candidate 2679.2559 2679.3798 -46.2 GAVQVFIMLLLATVSDCAVITGACER 2679.2559 2679.1846 26.6 SYDPPCPGHWTPEAPGSGTTCPGLPR 2679.2559 2679.2889 -12.3 DGETAEEQGGPVPPPVAPGGPGLGGAPGGR 2679.2559 2679.3286 -27.1 GAGALALGASEPCNLSSAAPLPDGAATAAR 2679.2559 2679.2010 20.5 AVDFQEAQSYADDNSLLFMETSAK 2679.2559 2679.3102 -20.3 GTYLPQTYIIQEEMVVTEHVSDK 2679.2559 2679.25575 0.1 ERPTPSLNNNC(carbamidomethyl)TTSEDSLVLYNR

… and many more…

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2 Tandem MS: Precursor Mass- based Search Compare theoretical product ions to experimental product Ions from MS2 spectrum

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 COMPARE 2 theoretical product ions (table below) to experimental product Ions from MS2 spectrum (matched ions are colored blue and red)

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Evaluate Spectrum for Peptide 2 Candidate Match Quality

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2 Tandem MS: Precursor Mass-based Search

SUMMARY- for Each MS/MS Spectrum: • Generate theoretical product ion tables for all peptide candidates • Compare theoretical product ions to experimental product Ions from MS2 spectrum • Score all candidate peptides • Rank peptides by Score

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 2 Tandem MS: Precursor Mass-based Search Find Theoretical Peptide Matches from in silico STEP 1 Protein Digest in the range: “Experimental Precursor Mass +/- Mass Tolerance”

For each candidate peptide from Step 1: Compare STEP 2 Theoretical Product Ions (b & y, etc) to Experimental Product ions (data)

SCORE: many software programs/ algorithms STEP 3 Peptide rank PROTEIN Report and Grouping: many variations

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Tandem MS: Precursor Mass- 2 based search Software at UM

• Sequest • X!Tandem • MASCOT

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Three Strategies for Protein Inference from Peptide MS or MS/MS Data

Peptide Mass Peptide MS/MS DATA INPUT: List (MS1) (MS2)

Peptide Mass Precursor Sequence- Search TYPE: Fingerprint Mass-based based (PMF) (historical) (de novo or 2 mass tag) 1 3

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3 MS/MS Sequence-based Software at UM • ProteinPilot® (AB Sciex) • PEAKS (BSI)

Open-source Software • Direct Tag (D Tabb Lab)

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3 Sequence-based Searches: WHEN?

• Unsequenced genome (protein(s) of interest are not in the database)  HOMOLOGY-based search • Search for amino acid MUTATIONS • Search for LARGE NUMBERS of Post Translational Modifications (PTM’s)

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3 Tandem MS: Sequence-based Search

STEP 1: Obtain [full or partial] amino acid sequence string directly from the spectrum by de novo Sequencing

de novo (Latin) "from the beginning"

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3 Tandem MS: Sequence-based Search STEP 1: de novo sequencing: amino acid sequence is determined from delta mass values for a series of successive peptide b- or y-type product ions from a Product Ion (MS/MS) spectrum

EXAMPLE de novo sequencing:

“delta masses” between L V A D L E peaks correspond to amino acid residue masses ∆113 ∆71 ∆115 ∆99 ∆113 ∆129

Center for Mass Spectrometry and ProteomicsSee amino acid | residuePhone masses: | (612)625 http://www.ionsource.com/Card/aatable/aatable.htm-2280 | (612)625-2279 3 Tandem MS: Sequence-based Search STEP 2: QUERY THE PROTEINS Example: AB Sciex Paragon® Algorithm For each MS/MS spectrum, multiple, short sequences (taglets) are generated, scored, and matched against protein sequences. “Sequence Temperature Values” (STV) rank candidate amino acid sequence segments. STV is related to the number of taglet hits per region.

Slide content: courtesy of Sean L. Seymour, AB Sciex

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3 Tandem MS: Sequence-based Search

STEP 3: Candidate peptide sequences within protein sequences are identified with Precursor mass AND sequence tag information Include: • Enzyme specificity • Mass tolerance • Amino acid modifications • Amino Acid mutations

STEP 4: Candidates are Ranked and SCORED

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 3 Tandem MS: Sequence-based Search STEP 3b: Error-tolerant Sequence Tag Reconciliation Includes Amino Acid Mutation Search Component

Combination: sequence tag & mass match

Slide content: adapted from David L. Tabb, Vanderbilt University: Bioinformatics 2009, Baltimore MD

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Search Algorithm Summary

1) Several different search algorithm types exist 2) Each program provides useful results - no unified method exists 3) The same data searched using different algorithms may yield different results (scoring and ranking schemes are distinct)

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

Thursday AM Lecture 3: Protein Validation and Quantitation

Candace Guerrero

• Statistics • P-value • Peptide scoring • Multiple hypothesis testing • Multiple hypotheses • False discovery rate • False discovery rate • Target / Decoy • Protein inference • One hit wonders • Shared peptide • Parsimony

346 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Statistics

Nature. 2014 Feb 13;506(7487):150-2

347 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Classical Example

Nature. 2014 Feb 13;506(7487):150-2

348 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Significance Testing

• Assumptions – Normal distribution – Independent – Scoring model • Null Hypothesis • Test statistic • P-value • Conclusion http://en.wikipedia.org/wiki/P-value

349 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide Spectrum Match Scoring

• Visual inspection • Scoring function f(S,P) measures the quality of the match between spectrum S and peptide sequence P

350 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide Spectrum Match Scoring

• Look at fragment ions – predicted mass – expected intensity – compare to next best – presence of immonium ions – etc.

351 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MULTIPLE HYPOTHESIS TESTING & FALSE DISCOVERY RATE

352 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Multiple Hypothesis Testing Errors

The problem of multiplicity arises from the fact that as we increase the number of hypotheses in a test, we also increase the likelihood of witnessing a rare event, and therefore, the chance to reject the null hypotheses when it's true (Type I error).

353 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Common Statistical Measures

Truth

H0 True H0 False

Correct Type II Error (β) H True 0 (TP) (FP)

Type I Error (α) Correct H0 False Your Findings Your (FN) (TN)

True Positive Rate: TP / (TP + FN) False Positive Rate: FP / (FP + TN)

354 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Multiple Hypothesis Testing Correction

• Bonferroni – Simplest correction: Bonferroni correction – divide by number of tests. • Example – If you want final error rate of 5% and you have 1000 proteins (1000 hypothesis tests), then divide the single test P-value by 1000, giving you P = 0.00005 as the threshold you would apply to have a 5% error rate in the proteins that pass this threshold – Too stringent – want to accept more errors

355 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Multiple Hypothesis Testing Correction

• False Discovery Rate (FDR) – Benjamini & Hochberg, 1994 – Controls for the expected proportion of falsely rejected hypotheses • Requires we know false identifications • Decoy database – Reversed or random sequences

356 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Common Statistical Measures

Truth

H0 True H0 False

Correct Type II Error (β) H True 0 (TP) (FP)

Type I Error (α) Correct H0 False Your Findings Your (FN) (TN)

False Discovery Rate: FP / (FP + TP) True Positive Rate: TP / (TP + FN) False Positive Rate: FP / (FP + TN)

357 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Target (Forward) / Decoy (Reverse)

>gi|6319436|ref|NP_009518.1| Pol12p [Saccharomyces cerevisiae S288c] MSGSIDVITHFGPDADKPEIITALENLTKLHALSVEDLYIKWEQFSNQRRQTHTDLTSKNIDEFKQFLQLQMEK RANQISSSSKVNTSTKKPVIKKSLNSSPLFGLSIPKTPTLKKRKLHGPFSLSDSKQTYNVGSEAETNEKGNSSL F KLEFTPGMAEDAVGDSAPLSHAKSSDAKTPGSSTFQTPTTNTPTTSRQNVPAGEILDSLNPENIEISSGNPNV GLLSTEEPSYNQVKVEPFYDAKKYKFRTMRQNLQEASDVLDDQIESFTKIIQNHYKLSPNDFADPTIQSQSEIY AVGRIVPDSPTYDKFLNPESLSLETSRMGGVGRRVRLDLSQVNELSFFLGQIVAFKGKNANGDYFTVNSILPL PYPNSPVSTSQELQEFQANLEGSSLKVIVTCGPYFANDNFSLELLQEFIDSINNEVKPHVLIMFGPFIDITHPLIA SGKLPNFPQFKTQPKTLDELFLKLFTPILKTISPHIQTVLIPSTKDAISNHAAYPQASLIRKALQLPKRNFKCMAN PSSFQINEIYFGCSNVDTFKDLKEVIKGGTTSSRYRLDRVSEHILQRRYYPIFPGSIRTRIKPKDVSTKKETNDM ESKEEKVYEHISGADLDVSYLGLTEFVGGFSPDIMIIPSELQHFARVVQNVVVINPGRFIRATGNRGSYAQITVQ CPDLEDGKLTLVEGEEPVYLHNVWKRARV DLIAS

###REV###>gi|6319436|ref|NP_009518.1| Pol12p [Saccharomyces cerevisiae S288c] SAILDVRARKWVNHLYVPEEGEVLTLKGDELDPCQVTIQAYSGRNGTARIFRGPNIVVVNQVVRAFHQLESPII MIDPSFGGVFETLGLYSVDLDAGSIHEYVKEEKSEMDNTEKKTSVDKPKIRTRISGPFIPYYRRQQLIHESVRD R LRYRSSTTGGKIVEKLDKFTDVNSCGFYIENIQFSSPNAMCKFNRKPLQLAKRILSAQPYAAHNSIADKTSPILV TQIHPSITKLIPTFLKLFLEDLTKPQTKFQPFNPLKGSAILPHTIDIFPGFMILVHPKVENNISDIFEQLLELSFNDN AFYPGCTVIVKLSSGELNAQFEQLEQSTSVPSNPYPLPLISNVTFYDGNANKGKFAVIQGLFFSLENVQSLDLR VRRGVGGMRSTELSLSEPNLFKDYTPSDPVIRGVAYIESQSQITPDAFDNPSLKYHNQIIKTFSEIQDDLVDSA EQLNQRMTRFKYKKADYFPEVKVQNYSPEETSLLGVNPNGSSIEINEPNLSDLIEGAPVNQRSTTPTNTTPTQ FTSSGPTKADSSKAHSLPASDGVADEAMGPTFELKLSSNGKENTEAESGVNYTQKSDSLSFPGHLKRKKLTP TKPISLGFLPSSNLSKKIVPKKTSTNVKSSSSIQNARKEMQLQLFQKFEDINKSTLDTHTQRRQNSFQEWKIYL DEVSLAHLKTLNELATIIEPKDADPGFHTIVDISGSM

Elias JE, Gygi SP. Nat Methods. 2007 Mar;4(3):207-14.

358 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Target / Decoy

F possibly correct

R clearly wrong

Elias JE, Gygi SP. Nat Methods. 2007 Mar;4(3):207-14.

359 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 FDR in Practice

1. Sort all peptide hits by score, descending. 2. Count how many target hits are greater than or equal to a given score 3. Count how many decoy hits are greater than or equal to a given score 4. Estimate the total number of incorrect hits (false positive, FP) from observed decoy hits (d) greater than or equal to a given score: FP=df 5. Calculate statistics related to FP for each given score threshold. 6. Select score threshold based on a desired statistic threshold.

Methods Mol Biol. 2010; 604: 55–71

360 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 # Spectrum Accession Peptide Score 1 scanJust 3632 P35908 to GFSSGSAVVSGGSRReview: 4.6 2 scan 3609 P0AFY8 FSAASQPAAPVTK 3.7 3 scan 3629 P0A940 GFQSNTIGPK 3.0 4 scan 3635 P0A6F9 STRGEVLAVGNGR 2.2 5 scan 3636 P0A870 ELAESEGAIER 2.1 6 scan 3607 P0A799 ADLNVPVKDGK 1.9 7 scan 3626 P0ABC7 EAEAYTNEVQPR 1.6 8 scan 3602 P0A853 IRVIEPVKR 1.4 9 scan 3623 P38489 KLTPEQAEQIK 0.9 10 scan 3616 P00448 GTTLQGDLK 0.8 11 scan 3621 P09546 LLPGPTGER 0.4 12 scan 3615 P0AFG8 AFLEGR 0.2 13 scan 3624 P14565 SAADVAIMK 0.0 14 scan 3613 rev_P06864 EGSLAVNVQGDAAIR -0.4 15 scan 3604 P36562 DPEEVVGIGANLPTDK -0.7 16 scan 3606 P0A9C5 IPVVSSPK -0.7 17 scan 3611 P0ABB0 ASTISNVVR -0.7

Peptide FDR Peptide 18 scan 3614 rev_Q2EEU2 KFVALTCDTLLLGER -0.8 19 scan 3620 rev_P0ACL5 NNESAALMKEYCR -0.9 20 scan 3633 rev_P37309 SDGSCNQRALNR -0.9 21 scan 3627 P32132 VEETEDADAFRVSGR -1.0 22 scan 3618 P37342 ILTQDEIDVR -1.0 23 scan 3610 rev_P0ADK0 IANVSDVVPR -1.2 24 scan 3601 P0AG93 LGMKREHMLQQK -1.3

361 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 # Spectrum Accession Peptide Score 1 scanJust 3632 P35908 to GFSSGSAVVSGGSRReview: 4.6 2 scan 3609 P0AFY8 FSAASQPAAPVTK 3.7 3 scan 3629 P0A940 GFQSNTIGPK 3.0 4 scan 3635 P0A6F9 STRGEVLAVGNGR 2.2 5 scan 3636 P0A870 ELAESEGAIER 2.1 6 scan 3607 P0A799 ADLNVPVKDGK 1.9 7 scan 3626 P0ABC7 EAEAYTNEVQPR 1.6 8 scan 3602 P0A853 IRVIEPVKR 1.4 9 scan 3623 P38489 KLTPEQAEQIK 0.9 10 scan 3616 P00448 GTTLQGDLK 0.8 11 scan 3621 P09546 LLPGPTGER 0.4 12 scan 3615 P0AFG8 AFLEGR 0.2 13 scan 3624 P14565 SAADVAIMK 0.0 14 scan 3613 rev_P06864 EGSLAVNVQGDAAIR -0.4 15 scan 3604 P36562 DPEEVVGIGANLPTDK -0.7 16 scan 3606 P0A9C5 IPVVSSPK -0.7 17 scan 3611 P0ABB0 ASTISNVVR -0.7

362 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 # Spectrum Accession Peptide Score 1 scanJust 3632 P35908 to GFSSGSAVVSGGSRReview: 4.6 2 scan 3609 P0AFY8 FSAASQPAAPVTK 3.7 3 scan 3629 P0A940 GFQSNTIGPK 3.0 4 scan 3635 P0A6F9 STRGEVLAVGNGR 2.2 5 scan 3636 P0A870 ELAESEGAIER 2.1 6 scan 3607 P0A799 ADLNVPVKDGK 1.9 7 scan 3626 P0ABC7 EAEAYTNEVQPR 1.6 8 scan 3602 P0A853 IRVIEPVKR 1.4 9 scan 3623 P38489 KLTPEQAEQIK 0.9 10 scan 3616 P00448 GTTLQGDLK 0.8 ? 11 scan 3621 P09546 LLPGPTGER 0.4 12 scan 3615 P0AFG8 AFLEGR 0.2 13 scan 3624 P14565 SAADVAIMK 0.0 14 scan 3613 rev_P06864 EGSLAVNVQGDAAIR -0.4 15 scan 3604 P36562 DPEEVVGIGANLPTDK -0.7 16 scan 3606 P0A9C5 IPVVSSPK -0.7 17 scan 3611 P0ABB0 ASTISNVVR -0.7

363 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 From peptides to proteins… PROTEIN INFERENCE

364 From Peptides 85% to Proteins AEPTIR

(4%) 65% Protein IDVCIVLLQHK

25% 0.15 * 0.35 * 0.75 = 0.04

Feng J, Naiman DQ, Cooper B. NTGDR Anal Chem. 2007 May 15;79(10):3901-11.

365 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide 1 Peptide 2 Correct Protein A Peptide 3 Peptide 4 Correct Protein B Peptide 5

Peptide 6 Incorrect Peptide 7 Protein C

Peptide 8 Incorrect Peptide 9 Protein D Peptide 10 80% Peptides 50% Proteins 366 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 One Hit Wonders

(5%) 95% Protein IDVCIVLLQHK

367 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 One Hit Wonders

• Quantify as a score: If different peptides agree: Good! If peptides are one-hit-wonders: Bad! • Protein Prophet, etc. • Then can use FDR at protein level

368 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 # Accession Protein Score 1 P0ABH7 4258.08 2 P0ABJ9 2423.84 3 P0A7S3 1670.86 4 P0ACF0 1230.35 5 P0AES0 896.12 6 P21165 702.89 7 P0AG59 524.04 8 P17952 409.74 9 P08997 327.85 10 rev_P76577 276.03 11 P41407 246.88 12 P39177 219.44 13 P37689 195.37 14 P0A951 177.02 15 P0AGG4 164.52 16 P29131 153.92 17 rev_P0AEQ1 146.86 18 rev_P09155 140.07 19 P0A9S5 132.29 20 P0AE45 125.41 21 P77718 120.12 22 P76115 116.15 23 rev_P76463 111.37 24 rev_P0A6E4 107.58

369 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 # Accession Protein Score 1 P0ABH7 4258.08 2 P0ABJ9 2423.84 3 P0A7S3 1670.86 4 P0ACF0 1230.35 5 P0AES0 896.12 6 P21165 702.89 7 P0AG59 524.04 8 P17952 409.74 9 P08997 327.85 10 rev_P76577 276.03 11 P41407 246.88 12 P39177 219.44 13 P37689 195.37 14 P0A951 177.02 15 P0AGG4 164.52 16 P29131 153.92 17 rev_P0AEQ1 146.86 18 rev_P09155 140.07 19 P0A9S5 132.29 20 P0AE45 125.41 21 P77718 120.12 22 P76115 116.15 23 rev_P76463 111.37 24 rev_P0A6E4 107.58

370 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Protein FDRs only accurate with >100 Proteins 12% 10% 8% 6% 4%

2% 1% Error In FDR Estimation

Uncertainty in Protein FDR in Protein Uncertainty 0% 0 200 400 600 Number of Confidently IDed Proteins

Nesvizhskii, A. I.; Aebersold, R. 373 Mol. Cell. Proteom. 4.10, 1419-1440, 2005

85% Tubulin alpha 6

YMACCLLYR Tubulin 85% alpha 3

Tubulin alpha 4 SIQFVDWCPTGFK 85% Shared Peptides Peptides Shared

374 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide / Protein Quantification

375 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Outline Terminology

• MS Quantification • Absolute Quantification • Why variance matters • Relative Quantification • Label-free / normalization • Bias • Labelled • Variability / Variance – iTRAQ • Label-free • Peptides to proteins • Normalization • Labelled • iTRAQ • Isobaric • Reporter ions

376 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Peptide / Protein Quantification

• Absolute – Estimate the molar amount of protein / peptide in the biological sample – PTMs – Validation • Relative –Fold change / statistically significant difference between 2 biological states – Biological variation – Biomarker studies

377 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Label-free

• Area Under Curve – MS1 – Integrate XIC

• Spectral Counting – MS2 – High abundant proteins

Käll L, Vitek O (2011) Computational Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277

378 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MS1 AUC

XIC for m/z = 421.76 y = f(x)

Area = ( ) 𝑏𝑏 𝑎𝑎 379 Center for Mass Spectrometry and Proteomics∫ | Phone𝑓𝑓 𝑥𝑥 | (612)625-2280 | (612)625-2279 Typical Discovery Experiment

Patient Group A Patient Group B Population Biological Prepare Samples

Peptides Instrument Sample HPLC-MS/MS Handling

MS1 & MS2 Spectra

Preprocess Sources of Quantify Identify Analyze Statistically Bias and Variability

Differentially Abundant Peptides/Proteins

380 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 MS Quantification

• MS not inherently quantitative • Physiochemical properties invoke different MS responses • MS only samples a small percentage of total peptides • Bias and variability

Population Biological

Instrument Sample Handling

381 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Why Variance Matters

382 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Normalization

• Remove bias and variability between runs • Global – commonly used – Median scale – Total ion current (TIC) • Local – very recent development – Proximity-based intensity normalization (PIN)

383 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Labeled Quantification

• Run samples simultaneously on in a single run • Add label to samples • Mix samples together • Compute ratios / statistically significant diffs.

384 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Labeled

• Isobaric – MS2, iTRAQ – Number of samples • Synthetic Peptides – MS1 – Absolute (AQUA) • Metabolic – MS1, SILAC – Not higher life forms Käll L, Vitek O (2011) Computational Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277

385 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 Isobaric Example - iTRAQ

www.moffitt.org

386 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 iTRAQ® 8-Plex Reagent Chemical Structure

Isobaric Tag Total mass = 305

Amine specific peptide O reactive group (NHS) ~~~~~~~ O Reporter Group N N N-hydroxysuccinimide 113 –119, 121 m/z N O

Balance Group (?) Mass 184, 186 – 192 m/z

N N N N 13 CH2

13 CH2 15N+ 15

m/z N+ N+ N+

13 13 CH2 CH2 113 114 115 116

13 13 13 CH3 CH3 CH3

15N 15 15 119, 121 119, N N N 13 13 13 13 13

– CH2 CH2 CH2 CH2 CH2

13 13 13 13 13 CH2 CH2 CH2 CH2 CH2 15N+ 15N+ 15N+ 15N+

113 Applied Biosystems has granted 13 13 13 CH2 CH2 CH2 permission to use this slide.

Isobaric Groups Reporter 117 118 119 121

387 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 iTRAQ Experiment

Normal Brain Normal Brain Huntington’s Huntington’s Tissue-1 Tissue-2 Disease-1 Disease-2 Obtain protein- containing sample, extract protein

Reduce, alkylate Reduce, alkylate Reduce, alkylate Reduce, alkylate Cysteines Cysteines Cysteines Cysteines

Proteolytic Digestion Trypsin Trypsin Trypsin Trypsin Digest Digest Digest Digest

Label peptides with iTRAQ TAG iTRAQ TAG iTRAQ TAG iTRAQ TAG iTRAQ® Reagents 114 115 116 117

MIX

2D LC-MS/MS Tissue Images: Rosas HD et al, (2002) Neurology, 58, 695

388 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

iTRAQ Experiment MS2 Spectrum

Peptide iTQVAIVVGAPR

1200 MW 1024.62

216.2 Peptidemono = VAIVVGAPR 255.1 ProteinMW Match = 1024.6249Platelet

1100 Protein ID = plateletmembrane membrane

145.1

b3 1000 glycoproteinglycoprotein 11b

11b 428.3

900

114.1 “Reporter Ion Mass Tags” from

800 which quantitation is

b1 y8

2) 9(+

y calculated 700

244.2 782.5

y1 600 513.3

0.2 40

11 7.1 b2

175.1 500

Intensity,counts

315.2

a4 Peptide match is made a5 y5

400 from product ions, e.g., b-

598.4 499.3 300 185.1

and y-ion seriesy9

212.1

y7 y2

200 I

881.6

711.4 I b4

272.2 I I I

y3 100 70.1

383.3

86.1 527.4

199.1

343.2

412.3

230.2 326.2

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 440.3

m/ z, amu

389 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 iTRAQ Results

• Reporter ion intensities reflect relative peptide amounts

control disease

100 115.13 976.9

90 114.13 116.13 117.13 80

40 %Intensity

No change disease:control 10 113.11

0 112.72493 113.91559 115.10626 116.29692 117.48759 118.67826 Mass (m/z)

390 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 iTRAQ Results

• What fold changes are significant?

• Do they represent biological relevance as opposed to experimental variability?

control disease

100 116.14 117.14 1.1E +4

%Intensity 115.14

Increase disease:control 30 114.14

10 113.12 0 112.74415 113.88269 115.02123 116.15977 117.29831 118.43685 Mass (m/z)

control disease

100 115.12 3070.1

90 114.12

60 116.12 50 117.12

40 %Intensity

Decrease disease:control 30

10 113.11

0 112.52129 113.84865 115.17602 116.50338 117.83075 119.15811 Mass (m/z)

391 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

free - Labeled & & Label Labeled From Käll L, Vitek O (2011) Computational Mass Spectrometry–Based Proteomics. PLoS Comput Biol 7(12): e1002277. doi:10.1371/journal.pcbi.1002277

392 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 From Peptides to Proteins APEX

Lu, et al., Nature Biotechnology 25, 117 - 124 (2007)

393 Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279 394 Appendix: Useful Links Introduction to Mass Spectrometry

University of Minnesota – Biodale Resources: https://cbs.umn.edu/research/resources/biodale

About Mass Spec: https://www.asms.org/about-mass-spectrometry

What is Electrospray? http://newobjective.com/electrospray/ Associations

American Society for Mass Spectrometry https://www.asms.org/

Association of Biomolecular Resource Facilities (ABRF) https://abrf.org/

Minnesota Chromatography Forum http://www.minnchrom.com/

Minnesota Mass Spec Discussion Group (MinnMass) http://minnmass.org/

HUPO https://hupo.org Human Proteome Organization

US HUPO www.ushupo.org Companies

SCIEX https://sciex.com/

Bruker Daltonics https://www.bruker.com/

Waters http://www.waters.com/

Thermo https://www.thermofisher.com

Cambridge Isotope Laboratories http://www.isotope.com/ Software & Search Tools

Bioinformatics Solutions (PEAKS software) http://www.bioinfor.com/

Proteome Software (Intro. to Proteomics and Scaffold software) https://proteomesoftware.zendesk.com/hc/en-us

Matrix Science (Mascot software) http://www.matrixscience.com/ mMass (free, open source software tool for MS) http://www.mmass.org/ MolE – Molecular Mass Calculator v2.02 http://mods.rna.albany.edu/masspec/MoIE

UCSF and Protein Prospector http://prospector.ucsf.edu/prospector/mshome.htm

Sequest home page at Scripps http://fields.scripps.edu/yates/wp/?page_id=17

Sequest Tips from Novatia https://www.enovatia.com/ms/ms-resources/sequest-tips/

XCMS Online at Scripps https://xcmsonline.scripps.edu/landing_page.php?pgcontent=mainPage Protein/Mass Spec Information

High Resolution Mass Calculator and Isotope Distribution Calculator http://www.sisweb.com/mstools/isotope.htm

DeltaMass (database of protein post translational modifications at ABRF) https://abrf.org/delta-mass

IonSource (mass spectrometry educational resource) www.ionsource.com

IUPAC Mass Spectrometry Definitions of terms http://www.iupac.org/publications/pac/85/7/1515/

NCBI Homepage https://www.ncbi.nlm.nih.gov/

UniMod (protein modifications for MS – login as guest) http://www.unimod.org/login.php?message=expired

UniProt Protein Database https://www.uniprot.org/

Common MS Contaminants (from NC State MSF) https://chemistry.sciences.ncsu.edu/msf/usefulMSFinfo.html

Protein Pilot Web Tutorial Modules https://netfiles.umn.edu/users/pjagtap/Proteomics_Webinars_at_MSI./ProteinPilot_Web_Modules.pdf IonSource Amino Acid Table

Return to IonSource.Com Home

AA Codes Mono. AA Codes Mono.

Gly G 57.021464 Asp D 115.02694

Ala A 71.037114 Gln Q 128.05858 I Ser S 87.032029 O Lys K 128.09496 N Pro 97.052764 S Glu E 129.04259 P O U Val 99.068414 R Met M 131.04048 V C E Thr T 101.04768 . His H 137.05891 C Cys C 103.00919 O Phe F 147.06841 M Leu L 113.08406 Arg R 156.10111

Ile I 113.08406 CMC 161.01467

Asn N 114.04293 Tyr Y 163.06333

- - - Trp W 186.07931

Proteomics Core Facility SAMPLE PREPARATION USER GUIDE

On the use of SDS-PAGE gels

For in-gel digestion of proteins, basically any type of SDS-PAGE gel will work (gradient or non-gradient, self-cast or precast, reducing or non-reducing), as long as you use high-grade reagents to prepare them, and fresh buﬀers to run them. If you use home-made gels, don’t cast and run them on the same day. Leaving a gel overnight will allow polymerization to complete, thus preventing crosslinking of proteins in the gel by residual radicals. This is best done at room temperature (not in the fridge/cold room) by covering the gel in plastic, adding some wet tissue to prevent drying of the gel. Gels can be kept at least a week before running. Please observe the shelf-life of precast gels.

Coomassie-stained gels

Not all coomassie stains are compatible with mass spectrometry. We strongly recommend that you mix your own coomassie stains (see protocol below for the recipe). Always try to only stain your gel for the minimum time to ( just) detect your protein(s) of interest. Remember that staining is only meant to visualize the band. Extended staining will increase the background in subsequent MS analysis, it will NOT increase the amount of protein. Destain the gel thoroughly to clear the background and to enhance visibility of the band. Do NOT heat your gel in the microwave to speed up the staining! This will bake your protein in the gel, we will never get it out again.

We DO NOT accept samples that have been stained with any commercial 'instant' or 'ready-to-use' staining kits. Please NEVER use overhead projector foils for gel scanning as this prevents the gel from being used for MS experiments.

A protocol for colloidal coomassie staining can be downloaded here.

Scanning of gel bands

It is very helpful to have a scan of the gel before processing it for MS analysis. To prevent contamination (see below), ﬁrst seal the gel in plastic (foil and sealer and scanner are available in the Facility, which you are welcome to use). Only use a dedicated lab-scanner that is not used for other purposes (e.g. in the oﬃce). A gel scanner is available in the Facility – feel free to use them. Do NOT use overhead transparencies for scanning – this WILL introduce contaminating polymers that inhibit protease activity.

Cutting of gel bands

We prefer to receive intact gels and do the cutting ourselves (if possible, include a scan appropriately indicating your bands of interest). Alternatively, you can cut bands yourself, but please observe these guidelines (also read the section below on ‘contamination’). After cutting, gel bands can be kept for months at -20°C before MS analysis without adverse eﬀect. For uniformity in sample preparation, it may be a good idea to prepare other bands from the same gel at the same time, even if you are not planning to send them now.

Contamination

Contaminants may be introduced at several steps during sample preparation. Some will go unnoticed, others will totally obscure all proteins. Therefore, minimizing contamination is essential.

Keratins

Keratins are omni-present and notorious contaminating proteins originating from skin, hair, dust, clothes, chemicals, etc. Total elimination is virtually impossible (and not necessary), but over-abundance will result in the repeated identification of keratins, and not your protein of interest. Most contaminating keratins are believed to originate from dust collected on the gel after running it. Therefore, a clean lab environment will certainly benefit the outcome of the MS experiment. If you don’t trust your lab is sufficiently dust-free, you are most welcome to run/cut your gel in our Facility. Alternatively, there are a couple of measures you can take. For starters, wear powder-free nitrile gloves at all times. Latex gloves often contain starch, and thus a lot of protein, so do not use these. Never touch the gel (or the liquid it is floating in) with bare hands. Use clean trays/containers that are only used for the purpose of gel staining (and not for e.g. blocking solutions for Western blots), and leave them closed with a lid during staining/storage. Don’t lean over gels, scratching your head how to interpret the band pattern. If you take the gel out of the container (e.g. for scanning, cutting), thoroughly clean the area with water (no soap!) or 70% ethanol. Always use fresh solutions to prepare and run gels, including sample loading buffer. Old solvents tend to collect dust – literally.

Other contaminating proteins

Other frequently observed proteins are BSA, immunoglobulins, and other serum proteins. These usually originate from cell culture media, and can be reduced by extensive washing of cell pellets.

Polymers

In a way, polymers are more serious contaminants than keratins, since they tend to stick to HPLC columns either ruining them, or at least causing a severe memory eﬀect in subsequent LC-MS runs. On top of this, most polymers ionize more easily than peptides - so that even minute amounts can be deleterious for the experiment. The most frequently observed polymers are various forms of PEG, which are present in some plastics but also in soaps, hand-creams (wear gloves!) and detergents. It is not always easy to trace back the source of a polymer contamination, but we have had issues with some brands of pipet tips (TopLab), and pipet tips that have been autoclaved in-house. Do not use coated (‘low-binding’) eppendorf tubes, and avoid using soft plastics (Paraﬁlm!). Furthermore, NO detergents should be present in samples to be submitted for MS, especially NP-40 and Triton X-100 have a bad reputation.

Reversed Phase Chromatography

In reversed phase chromatography peptides or proteins (or other molecules) partition between a hydrophobic stationary phase (e.g., C18 beads) and a less hydrophobic mobile phase (e.g., water or aqueous buffers). The partitioning of the sample components between the two phases is dependent on their respective solubility characteristics. Less hydrophobic components will spend more time in the mobile phase while more hydrophobic components will spend more time in the stationary phase.

Silica particles covered with chemically-bonded hydrocarbon chains (C2 to C18) form the hydrophobic stationary phase, while the less hydrophobic mobile phase is composed of an aqueous mixture of an organic solvent that surrounds the silica particles.

When a peptide or protein passes through a reversed phase column the partitioning mechanism operates continuously as the hydrophobicity of the aqueous mobile phase is increased by addition of an organic solvent such as acetonitrile.

Less hydrophobic peptides or proteins will always move faster than more hydrophobic ones, since the mobile phase is always less hydrophobic than the stationary phase.

Example: ZipTip® pipette tip

The ZipTip® pipette tip is a 10 uL pipette tip with a 0.6 uL bed typically packed with reversed phase (C18 or C4) chromatography media. It is intended for concentrating and purifying peptide, protein or oligonucleotide samples prior to MALDI or electrospray mass spectrometry.

Mass Definitions

Molecular masses are measured in Daltons (Da), or mass units (u).

One Dalton = 1/12 of the mass of a 12C atom.

Monoisotopic mass = sum of the exact masses of the most abundant isotope of each element present, i.e., 1H=1.007825, 12C=12.000000, 16O=15.994915, etc. The monoisotopic mass is the most accurately defined molecular mass and is preferred if a measurement of it can be determined.

Average mass = sum of the averaged masses (“isotope abundance weighted”) of the constituent elements of a given molecule.

The result is a weighted average over all of the naturally occurring isotopes present in the compound. This is the common chemical molecular weight that is used for stoichiometric calculations (H=1.0080, C=12.011, O=15.994, etc.). The average mass cannot be determined as accurately as the monoisotopic mass because of variations in natural isotopic abundances.

The mass to charge ratio (m/z). A quantity formed by dividing the mass (in u) of an ion by its charge number; unit: Thomson or Th

Isotopic Abundances of Common Elements*

Natural Abundance Element Isotope Mass (%) 1 H H 1.0078 99.99% 2 H 2.0141 0.015 12 C C 12 98.89 13 C 13.0034 1.11 By coincidence, the most 14 N N 14.0031 99.64 abundant isotope of 15 N 15.0001 0.36 these common elements 16 O O 15.9949 99.76 has the lowest mass. 17 O 16.9991 0.04 18 O 17.9992 0.2 31 P P 30.9737 100 32 S S 31.9721 95 33 S 32.9715 0.76 34 S 33.9679 4.22 36 S 35.9671 0.02 *A. Burlingame, UCSF

In-Gel Trypsin Digest Center for Mass Spectrometry & Proteomics, University of Minnesota (Adapted from EMBL Method)

• Wear gloves to minimize contamination from keratins. Do not lean, talk or breath over your gel when cutting bands. Do not have anyone walk by you when cutting out bands. Keratin is everywhere, so please take these precautions to minimize any exogenous keratin. • Siliconized tubes are highly recommended for proteolytic digestions. We prefer 1.5mL snap-cap Eppendorf Protein Lo-Bind microfuge tubes . • A typical gel band is ~2x10 mm(1mm thick). The volumes in this protocol correspond to gel volumes of this size. Please scale accordingly if your gel band/region is larger. If the gel band is smaller, the stated volumes are suitable. 1. Excision of protein bands from polyacrylamide gels. a) Excise the band of interest using a sterile scalpel, razor blade or sharpened microspatula. Cut as close to the protein band as possible to reduce the amount of “background” gel. Cut into ~2 x 2 mm cubes and place in 1.5 ml snap-cap microfuge tube. If cutting multiple bands, wash cutting utensil with 1:1 water/ethanol in between each gel band. b) If silver stain was used, destain bands with the protocol below; otherwise continue to step 2: Silver Destaining i. Excise the spot/band and place in very clean water (such as Milli-Q or nanopure water) in a siliconized microcentrifuge tube. Discard water. ii. De-stain (remove silver stain) gel piece with 1:1 solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulphate made FRESHLY before use. Use 300 – 500 uL, incubate for 8 minutes, and discard. iii. Add 100 uL of the destaining solution if necessary, incubate for 1 minute and discard. iv. Wash 4 x with 1 mL of Mill-Q water, 8 minutes per wash 2. Washing of gel pieces a) Transfer 75 ul 1:1 100mM ammonium bicarbonate:acetonitrile to gel pieces, vortex briefly and incubate 15 min at room temperature. Remove solution, discard to waste and repeat this step for a total of two washes. If the band is heavily Coomassie stained, an extra wash is recommended. b) Remove previous wash and add 75 ul of 100% acetonitrile. After pieces shrink and turn white and semi- opaque, remove acetonitrile (~30 sec – 1 min). c) Remove acetonitrile and proceed to step 3.

3. Reduction & Alkylation - *note that DTT and iodoacetamide solutions should be made fresh for each digest. a) Rehydrate gel pieces with 75 ul 10 mM DTT in 50 mM ammonium bicarbonate. Incubate 1 hr at 56 °C in water bath. After incubation, briefly spin down tubes then remove DTT solution. b) Add 75ul of 55 mM iodoacetamide in 50 mM NH4HCO3 and incubate 30 min at room temperature in the dark (iodoacetamide is light sensitive). Remove iodoacetamide solution. c) Wash gel plugs with 75 ul 1:1 acetonitrile:100 mM ammonium bicarbonate as in step 2. Repeat for a total of 2 washes. All the Coomassie should be removed at this time. If residual Coomassie remains, repeat wash with the acetonitrile:100 ammonium bicarbonate solution. d) Remove previous wash and add 75 ul of 100% acetonitrile. After pieces shrink and turn opaque white, remove acetonitrile (~30 sec-1 min). 4. In-Gel Digest a) Gel pieces should have the opaque white look from the last wash with 100% acetronitrile. b) Rehydrate gel pieces in digestion buffer at 4 °C (50 mM NH4HCO3, 5 mM CaCl2, 5 ng/ul trypsin). Add a sufficient volume of buffer to cover gel pieces, ~20 ul, inspect visually. Add more if necessary. c) Set on ice for 15 min. Remove supernatant and replace with 70 ul 50 mM NH4HCO3, 5 mM CaCl2. Incubate at 37 °C overnight in a warm-air incubator.

5. Extraction of Peptides a) Briefly spin down tubes in a centrifuge at low speed and recover supernatant from overnight incubation. Place supernatant in new 1.5 ml tube labeled accordingly. b) Add sufficient volume of 50% acetonitrile, 0.3% formic acid to cover gel pieces (approximately 60 ul), incubate 15 min. c) Recover supernatant and place in corresponding tube. d) Add the same volume used in step (b) of 80% acetonitrile, 0.3% formic acid, incubate 15 min. Recover supernatant and place in corresponding tube. e) Freeze pooled extracts in -80 °C freezer 30 minutes, then dry in speed vac. f) Proceed to zip tip/stage tip protocol (for desalting) or store at -80 °C prior to submission for mass spec analysis. Peptide Mass Fingerprint Database Search Parameters

• 1 or 0 Enzyme missed cleave sites (try each setting) • Database NCBI • Taxonomy (select appropriate, based on protein/sample origin) • Mass values MH+ • Peptide mass error tolerance 150 parts per million (ppm) (or, try 200 ppm alternatively) • Monoisotopic • Fixed modifications: carboxymethyl cysteine or carbamidomethyl cysteine (based on protein source) • Variable modification: oxidized methionine

Protein Digest Sample Species Taxonomy Acid glycoprotein Human Mammalian Alcohol dehydrogenase Yeast Yeast or Saccharomyces cerevisiae Aldolase Rabbit Mammalian Apotransferrin Rat Mammalian Bovine serum albumin Bovine Mammalian Cytochrome C Horse Mammalian Enolase Yeast Yeast or Saccharomyces cerevisiae Myoglobin Horse Mammalian