O HCD O TMT Reagent Generic Chemical StructureO N A. O O N N H

ETD NH2 Reactive Mass Group Mass Normalizer Reporter

O 10 ) O O N O B. TMT10plex Reagents (TMT * * * O N N * O * H 126 O 127C O N * O O * * O N * * N O H O N O * O * * O O 128C *N N * N * H O 127N O O * * O N * N O H O N O * * O * * O O 129C *N N * N * H O O 128N O * O N * * N O H * O N O O * * O O 130C *N N * N * H O * O 129N O * O * N N O * H * O N O * Thermo ScientificO Pierce Reagents N N O * * * Hfor Quantitative Proteomics 130N * O O N * * O O N N * H 131 * *

protein quantitation using mass spectrometry

Specialized reagents for discovery and targeted analysis table of contents

Overview 1

Discovery Quantitation Introduction 2-3 Discovery Quantitation Workflow by Sample Type 3 Protein Quantitation Reagents 4-19 SILAC Protein Quantitation Reagents and Kits 4-7 Isobaric Mass Tag Overview 8 Amine-reactive Tandem Mass Tag Reagents 9-12 (TMT Reagents) Cysteine-reactive Tandem Mass Tag Reagents 13-16 (iodoTMT Reagents) Carbonyl-reactive Tandem Mass Tag Reagents 17-19 (aminoxyTMT Reagents)

Targeted Quantitation

Introduction 20-21 Targeted Quantitation Workflow by Sample Type 21

Protein Quantitation Reagents 22-31 HeavyPeptide AQUA Standards 22-23 HeavyPeptide FasTrack Service 24-25 PEPotec SRM Peptide Libraries 26-27 Pierce Peptide Retention Time Calibration Mixture 28-29 Pierce 1-Step Heavy Protein In Vitro Expression Kit 30-31

Mass Spectrometry Systems and Capabilities 32 Related Products 33 References Back cover proteomics discovery vs. targeted analysis

an overview

A successful proteomics experiment requires For example, if the scope of a project is increased, it is understood that it the integration of the right sample preparation, will take more time or cost more money. Similarly, proteomics researchers must recognize the conflict of scalability, sensitivity and comprehensive instrumentation and software. In addition to analysis. It is impossible to achieve all three simultaneously. Strategies to these tools, a proteomics scientist also needs increase sensitivity and comprehensiveness generally require large sample the right strategy to achieve the intended quantities and multi-dimensional fractionation, which sacrifices throughput. Alternatively, efforts to improve the sensitivity and throughput of protein goals. Project managers are familiar with the quantification necessarily limit the number of features that can be monitored. conflicts of time, cost and scope; it is impossible For this reason, proteomics research is typically divided into two categories: to improve one of these without affecting discovery and targeted analysis. Discovery proteomics maximizes protein identification by spending more time and effort per sample and reducing the others. the number of samples analyzed. In contrast, targeted proteomics strategies limit the number of features that will be monitored, and then optimize the chromatography, instrument tuning and acquisition methods to achieve the highest sensitivity and throughput for hundreds or thousands of samples.

SENSITIVE

Discovery proteomics Targeted proteomics strategies optimize sensitivity strategies optimize sensitivity and and comprehensiveness scalability by limiting the number with few samples of monitored features THE PROTEOMICS CONFLICT

COMPREHENSIVE SCALABLE

It is impossible to optimize sensitivity, throughput and comprehensiveness simultaneously

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Measuring changes in global protein expression

These strategies reduce the dynamic range between components in an individual sample and reduce the competition between proteins or peptides for ionization and mass spectrometry (MS) duty cycle time. Quantitative discovery proteomics experiments add a further challenge because they seek to identify and quantify protein levels across multiple samples. Quantitative discovery proteomics experiments utilize label-free or stable isotope labeling methods to quantify proteins. Label-free strategies require highly reproducible fractionation, increased instrumentation time and alignment of peptides across LC-MS/MS experiments to compare spectral counts or ion intensities. Stable isotope protein labeling strategies (e.g., SILAC and Thermo Scientific™ Tandem Mass Tag™ methods) incorporate 2H, 13C, 15N or 18O isotopes into proteins and peptides, resulting in distinct mass shifts but otherwise identical chemical properties. This allows two to 10 samples to be labeled and combined prior to processing and LC-MS/MS analysis. Multiplexing reduces sample processing variability, improves specificity by quantifying the proteins from each condition simultaneously and requires less LC-MS and data analysis time. Quantitative proteomic studies are typically performed on high-resolution hybrid mass spectrometers, such as the Thermo Scientific™ Orbitrap™ Fusion™ Tribrid™, Thermo Scientific™ Orbitrap Elite™ and Thermo Scientific™ Q Exactive™ Mass Discovery proteomics experiments are intended Spectrometers. to identify as many proteins as possible across Specialized software including Thermo Scientific™ Proteome Discoverer™ 1.4, a broad dynamic range. This often requires SIEVE™ and ProSightPC™ software ensures as much high-quality data depletion of highly abundant proteins, is acquired, and as much valuable information is extracted from that data, as possible. enrichment of relevant proteins (e.g., protein immunoprecipitation) and fractionation steps (e.g., SDS-PAGE or chromatography) to decrease sample complexity.

2 For more information, or to download product instructions, visit thermoscientific.com/pierce Discovery Quantitation Workflow by Sample Type Using Thermo Scientific™ Products

Serum/Plasma Mammalian Cells Tissue Biofluids

• Metabolic Labeling Kits • Abundant Protein Depletion (SILAC Quantitation Reagents Reagents (Albumin, Top 2 or Top 12 Protein and Kits) Depletion Spin Columns and Kits) • Lysis & Fractionation Reagents (Mass Spec Sample Prep Kit for Cultured Cells)

• Protein Labeling using Active Site Probes (Kinase, GTPase and Serine Hydrolase Labeling and Enrichment Kits) • Protein Labeling of Cysteine-Reactive Sites (iodoTMT Isobaric Labeling Reagents and Kits)

• Protein Clean-Up (Desalting Spin Columns) • Protein Quantitation Reagents (BCA and Micro BCA Protein Assay Kits)

• Protein Digestion Verification (Digestion Indicator for Mass Spectrometry) • Protein Digestion - Proteases (Trypsin, LysC, LysN, Chymotrypsin, AspN, and/or GluC Proteases, MS Grade; In-gel Tryptic Digestion Kit; In Solution Tryptic Digestion and Guanidination Kit)

• Glycan Labeling of Reducing Sugars After PNGase Digestion (aminoxyTMT Isobaric Labeling Reagents) • Peptide Labeling of Amine-Reactive Sites (TMT Isobaric Labeling Reagents and Kits)

• Peptide Enrichment (Fe-NTA and/or TiO2 Phosphopeptide Enrichment Columns and Kits)

• Peptide Clean-Up (Detergent Removal Spin Columns) • Peptide Clean-Up (C18, Graphite Spin Columns and/or Tips)

• Thermo Scientific™ LTQ Orbitrap™ Velos™ Mass Spectrometer • Thermo Scientific™ LTQ Orbitrap™ Elite Mass Spectrometer Instruments • Orbitrap Fusion Tribrid Mass Spectrometer • Q Exactive Mass Spectrometer

• Proteome Discoverer 1.4 Software Software • SIEVE Software • Prosight PC Software

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Measuring changes in global protein expression

Thermo Scientific SILAC Protein Quantitation Kits and Reagents Complete kits for stable isotope labeling with amino acids in cell culture (SILAC).

Stable isotope labeling using amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. The SILAC method uses in vivo metabolic incorporation of “heavy” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry (MS) analysis for accelerated comprehensive identification, characterization and quantitation of proteins. Highlights: • Efficient – 100% label incorporation into proteins of living cells • Reproducible – eliminates intra-experimental variability caused by differential sample preparation • Flexible – media deficient in both L-lysine and L-arginine, allowing for more complete proteome coverage through dual amino acid isotope labeling • Versatile – broadest portfolio of liquid and powdered SILAC media based on classical media formulations, including DMEM, RPMI-1640, MEM, DMEM:F12, IMDM, Ham’s F12 and McCoy’s 5a Cells Grown in Light Cells Grown in Heavy • Compatible – label proteins expressed in a wide variety of mammalian cell Isotope-containing Media Isotope-containing Media + Treatment lines, including HeLa, 293T, COS7, U2OS, A549, NIH 3T3, Jurkat and others

Applications: • Quantitative analysis of relative changes in protein abundance from different Harvest & Lyse Cells cell treatments

• Quantitative analysis of proteins for which there are no antibodies available Quantitate Extracted Protein • Protein expression profiling of normal cells vs. disease states Mix Lysates • Identification and quantification of hundreds to thousands of proteins in a single experiment

• Simultaneous immunoprecipitation of labeled native proteins and protein Excise Bands complexes from multiple conditions

Trypsin Digestion

LC-MS/MS

Ratio Determination SDS-PAGE

Relative Intensity m/z Light Heavy

Figure 1. Schematic of SILAC workflow. A549 cells adapted to DMEM were grown for six 13 passages (10 days) using SILAC DMEM (Product # 89983) containing 0.1mg/mL heavy C6 L-lysine-2HCl or light L-lysine-HCl supplemented with 10% dialyzed FBS. After 100% label 13 incorporation, C6 L-lysine-labeled cells were treated with 5µM camptothecin (Sigma, St. Louis, Product # C9911) for 24 hours. Cells from each sample (light and heavy) were lysed using Thermo Scientific™ M-PER™ Mammalian Protein Extraction Reagent (Product # 78501). Samples were normalized for protein concentration using the Thermo Scientific™ Pierce™ BCA Protein Assay (Product # 23225), and 50mg of each sample were equally mixed before 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Gels were stained with Thermo Scientific™ GelCode™ Blue Stain Reagent (Product # 24592) and proteins were digested and alkylated using the Thermo Scientific™ Pierce™ In-Gel Tryptic Digestion Kit (Product # 89871) before analysis using a Thermo Scientific™ LTQ Orbitrap™ Mass Spectrometer.

4 For more information, or to download product instructions, visit thermoscientific.com/pierce SILAC requires growing mammalian cells in specialized media supplemented Example Experiment 12 13 with light or heavy forms of essential amino acids; e.g., C6 and C6 L-lysine, Using a SILAC Protein Quantitation Kit, A549 cells adapted to grow in Dulbecco’s 13 respectively. A typical experiment involves growing one cell population in medium Modified Eagle Medium (DMEM) were labeled with C6 L-lysine to > 98% containing light amino acids (control), while the other population is grown in the isotope incorporation. Heavy-labeled cells treated with camptothecin were lysed, presence of heavy amino acids (experimental). The heavy and light amino acids mixed with control lysates, separated by SDS-PAGE and digested with trypsin are incorporated into proteins through natural cellular protein synthesis. After before MS analysis. More than 350 proteins were successfully identified and alteration of the proteome in one sample through chemical treatment or genetic quantified using an LTQ Orbitrap Mass Spectrometer. manipulation, equal amounts of protein from both cell populations are then Most of the proteins identified had no change in abundance level after combined, separated by SDS-polyacrylamide gel electrophoresis and digested camptothecin treatment; however, 20% of proteins quantified in heavy-labeled with trypsin before MS analysis. Because peptides labeled with heavy and light cells had protein levels (SILAC ratios) 1.5-fold higher than control cells. One amino acids are chemically identical, they co-elute during reverse-phase column protein that was identified as being up-regulated 2.1-fold in response to prefractionation and, therefore, are detected simultaneously during MS analysis. camptothecin treatment was proliferating cell nuclear antigen (PCNA), a protein The relative peak intensities of multiple isotopically distinct peptides from each with involvement in DNA repair (Figure 2). To validate SILAC data, protein levels protein are then used to determine the average change in protein abundance in were separately quantitated by Western blot (Figure 3). In addition to an increase the treated sample (Figure 2). in PCNA protein levels, Capthesin-L protein abundance was decreased by 4-fold, Three different SILAC Kits are available, providing media that are compatible 14-3-3σ protein was increased 3.1-fold and glyceraldehyde-3-phosphate with different mammalian cell lines. Each kit includes all necessary reagents to dehydrogenase (GAPDH) protein did not significantly change (Figure 4). isotopically label cells, including media, heavy and light amino acid pairs, and The abundance ratios determined by Western blot were comparable to those dialyzed serum. Several isotopes of lysine and arginine are available separately, determined by SILAC. enabling multiplexed experiments and analysis. In addition, dialyzed FBS and other stand-alone media are available for additional mammalian cell lines. When combined with Thermo Scientific™ Protein/Peptide Sample Enrichment Products, Thermo Scientific™ SILAC Protein Quantitation Kits also enable MS analysis of low-abundance proteins such as cell-surface proteins, organelle-specific proteins and protein post-translational modifications such as phosphorylation or glycosylation. 1041.017 100 1041.518

90 H:L

80 PCNA 1.9 70 SILAC Ratio 1038.508 H:L = 2.1 60 1038.007 1042.020 50 p53 5.9 40 Relative Abundance 1039.010 30 1042.521 β-actin 1.2 20 1039.512 10 1043.023 GAPDH 1.1 0 Light (L) Heavy (H) 1036 1037 1038 1039 1040 1041 1042 1043 1044 1045 + m/z - Light (L) Heavy (H) 5µM Camptothecin

13 Figure 2. Representative MS spectra generated using SILAC. Light and heavy ( C6) Figure 3. Comparison of A549 protein levels detected by Western blotting after L-lysine-containing peptides (AEDNADTLALVFEAPNQEK) from PCNA were analyzed by MS. camptothecin treatment. Ten micrograms of each light (L) and heavy (H) sample were 13 Mass spectra of heavy peptides containing C6 L-lysine have an increased mass of 6Da and analyzed by 4-20% SDS-PAGE and Western blotting using specific antibodies. are shifted to the right of light peptide spectra by a mass to charge ratio (m/z) of 3 caused by a +2 ionization of peptides.

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 5 discovery quantitation

Measuring changes in global protein expression

A. B. 1197.14355 1090.49447 z=2 z=2 Capthesin L 100 100 14-3-3 1090.99583 SILAC Ratio 95 z=2 95 (AVATVGPISVAIDAGHESFLFYK*) (TTFDEAMADLHTLSEDSYK*) H:L = 3.10 90 90 85 85 SILAC Ratio 80 80 H:L = 0.25 75 75 70 70 65 65 60 1196.64294 60 z=2 55 55 1091.49703 50 50 z=2

Abundance 45 45 1197.64319 z=2 40 40 1200.15149 35 z=2 35 1087.98502 Relative Abundance Relative 1201.15186 z=2 30 30 1198.14587 z=2 25 1087.48574 1091.99823 25 z=2 1201.56921 z=2 1088.48745 z=2 20 z=2 20 z=2 15 15 1089.59712 z=2 1092.50363 10 10 1094.00011 1095.53064 1085.07452 1087.09293 z=2 z=2 z=3 5 5 z=2 z=? 0 0 1196 1197 1198 1199 1200 1201 1202 1086 1088 1090 1092 1094 1096 m/z m/z Light (L) Heavy (H) Light (L) Heavy (H)

C.

868.13474 z=3 100 GAPDH 95 866.12850 (VIHDNFGIVEGLMTTVHAITATQK*) z=3 90 85 867.80068 868.46861 SILAC Ratio 80 z=3 z=3 H:L = 1.12 75 866.46227 z=3 70 865.79440 65 z=3

60 55 50 45 866.79630 868.80253 40 z=3 z=3 35 Relative Abundance 30 25 20 867.13060 869.13672 z=3 15 z=3 10 869.47095 5 z=3 0 864 865 866 867 868 869 870 871 m/z Light (L) Heavy (H)

Figure 4. Identification and quantitation of Capthesin L, 14-3-3σ and GAPDH using SILAC. A. Representative MS spectra of light and heavy L-lysine containing peptide (AVATVGPISVAIDAGHESFLFYK) of Cathepsin L precursor. B. Representative MS spectra of light and heavy L-lysine containing peptide (TTFDEAMADLHTLSEDSYK) of 14-3-3σ. C. Representative MS spectra of light and heavy L-lysine containing peptide (VIHDNFGIVEGLMTTVHAITATQK) of 13 glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Mass spectra of heavy peptides containing C6 L-lysine (K*) have an increased mass of 6Da and are shifted to the right of light peptide spectra by a mass to charge ratio (m/z) of 3 caused by a +2 ionization of peptides.

6 For more information, or to download product instructions, visit thermoscientific.com/pierce Ordering Information

Product # Description Pkg. Size Product # Description Pkg. Size 13 89982 SILAC Protein Quantitation Kit – RPMI 1640 Kit 88436 C6 L-Leucine 500mg

Includes: RPMI Media for SILAC 2 x 500mL 88211 L-Proline 115mg Dialyzed FBS 2 x 50mL 13 C6 L-Lysine•2HCl 50mg 88430 L-Proline 500mg L-Lysine•2HCl 50mg 89984 RPMI Media for SILAC 500mL L-Arginine•HCl 2 x 50mg (RPMI-1640 minus L-Lysine and L-Arginine) 89983 SILAC Protein Quantitation Kit – DMEM Kit 88421 RPMI Media for SILAC 6 x 500mL Includes: DMEM Media for SILAC 2 x 500mL (RPMI-1640 minus L-Lysine and L-Arginine) Dialyzed FBS 2 x 50mL 13 C6 L-Lysine•2HCl 50mg 88426 Powdered RPMI Media for SILAC 104g L-Lysine•2HCl 50mg (RPMI minus L-Leucine, L-Lysine and L-Arginine) L-Arginine•HCl 2 x 50mg Sufficient for preparing 10L medium 88439 SILAC Protein Quantitation Kit - DMEM:F12 Kit 89985 DMEM Media for SILAC 500mL Includes: DMEM:F12 Media for SILAC 2 x 500mL (DMEM minus L-Lysine and L-Arginine) Dialyzed FBS 2 x 50mL 88420 DMEM Media for SILAC 6 x 500mL 13C L-Lysine•2HCl 50mg 6 (DMEM minus L-Lysine and L-Arginine) L-Lysine•2HCl 50mg L-Arginine•HCl 2 x 50mg 88425 Powdered DMEM Media for SILAC 135g (DMEM minus L-Leucine, L-Lysine and L-Arginine) 89989 L-Arginine•HCl 50mg Sufficient for preparing 10L medium 88427 L-Arginine•HCl 500mg 88422 MEM for SILAC 500mL 13 88210 C6 L-Arginine•HCl 50mg (MEM minus L-Lysine and L-Arginine) 13 88433 C6 L-Arginine•HCl 500mg 88214 Phenol Red Free MEM for SILAC 500mL 13 15 (MEM minus phenol red, L-Lysine and L-Arginine) 89990 C6 N4 L-Arginine•HCl 50mg 13 15 88215 DMEM:F12 (1:1) Media for SILAC 500mL 88434 C6 N4 L-Arginine•HCl 500mg DMEM:F12 (1:1) minus L-Lysine and L-Arginine 89987 L-Lysine•2HCl 50mg for induced pluripotent cells 88429 L-Lysine•2HCl 500mg 88424 Ham's F12 for SILAC 500mL 13 Ham’s F12 minus L-Lysine and L-Arginine) 89988 C6 L-Lysine•2HCl 50mg 13 88441 McCoy’s 5A Media for SILAC 500mL 88431 C6 L-Lysine-2HCl 500mg 13 15 88423 IMDM for SILAC 500mL 88209 C6 N2 L-Lysine•2HCl 50mg (IMDM minus L-Lysine and L-Arginine) 13 15 88432 C6 N2 L-Lysine•2HCl 500mg 89986 Dialyzed FBS for SILAC 50mL 88437 4,4,5,5-D4 L-Lysine•2HCl 50mg 88212 Dialyzed FBS for SILAC 100mL 88438 4,4,5,5-D4 L-Lysine•2HCl 500mg 88440 Dialyzed FBS for SILAC 500mL 88428 L-Leucine 500mg 13 88435 C6 L-Leucine 50mg

For a list of references using SILAC Reagents, please see the back cover.

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Measuring changes in global protein expression

Isobaric Mass Tagging Overview Simultaneously identify and quantify protein expression and post-translational modifications from multiple conditions in a single analysis.

Isobaric chemical tags are powerful tools that enable concurrent identification and quantitation of proteins in different samples using tandem mass spectrometry. These tags contain reactive groups that covalently label peptide amino termini, peptide cysteine amino acid side changes or glycopeptides, depending on the chemistry used. During the tandem mass spectrometry (MS/MS) analysis, the isobaric tag produces a unique reporter ion signature that makes quantitation possible. In the first MS analysis, the labeled peptides are indistinguishable from each other; however, in the tandem MS mode during which peptides are isolated and fragmented, the tag generates a unique reporter ion. Protein quantitation is then accomplished by comparing the intensities of the six reporter ions in the MS/MS spectra. The ability to generate low-m/z reporter ions and to distinguish them from isobaric interferences is essential for consistent, precise tandem mass tag quantitation. This is best accomplished using HCD fragmentation combined with the high- resolution-at-low-m/z detection that is available on Orbitrap-based systems.

A. Amine-reactive B. Sulfhydryl-reactive C. Carbonyl-reactive aminoxyTMTzero Reagent A. TMT Reagent Generic Chemical Structure Thermo Scientific iodoTMTzero Reagent Conjugation Reaction CID HCD HCD O O O O O O O H Cysteine R’ N N N N N NH2 N O N N I N N O H 126Da H H 126Da H NH H O O HS ETD ETD + ETD O Mass Mass Amine-Reactive Mass Mass Cys-Reactive Mass R Mass Carbonyl-Reactive Reporter Normalizer Group Reporter Normalizer Group Reporter Normalizer Group

R’ O O Figure 1. Structural design of Thermo Scientific Tandem Mass Tag Reagents. Mass reporter: Has a unique H N N NH mass and reports sample-specific abundance of a labeled peptide during MS/MS analysis.Cleavable linker:N N S H H Preferentially fragments under typical MS/MS conditions to release the mass reporter. Mass normalizer: Has O O a unique mass that balances the mass reporter, ensuring the same overall mass for all tags in a set. Reactive R A. TMT10plex Reagents (TMT10) B. TMTsixplex Reagents (TMT6) groups: A. Reactive NHS ester provides high-efficiency, amine-specific labeling of proteins/peptides.B. Reactive O iodoacetyl functional group provides covalent, irreversible labelingO of sulfhydryl (-SH) *groups. C. ReactiveO O O O O O ThermoO Scientific iodoTMTsixplex Reagents alkoxyamine functional group provides covalent labeling of carbonyl-containing compounds.* 126 N * * N N * * N N N * N * * O 126 * N * * O 129 * N * O * H H O HO O O O O * H O H * N N N * * N O O O * N ** * N O I 129 * N * N I 126 *H H H H O O O O O O O O O * O 127N *N N N * N 127C *N N N * N * N * * * O * N * * * O * N * * * O * N O H H 127 H 130 H * O O O * O O O * O O H H O * N N N * N * O * O * N ** * * N O I * N N I O O O O O O 127 *H O H O 130 H H O * O 128N *N * * N N * N 128C N N *N N N * * O * N * O * N* * O * N O H H 128 H 131 H O O O * O * * * * O O * O O H H * O * O N N * N N O O O O * N* * N I * N N I 128 H H 131 H H *N N N * N O O 129N * N * O * * N O 129C * * H H * O * O * O * O O O * O O 130N *N N N * N * * N O * N O 130C H H * O * O * O * O O 8 For more information, or to download product instructions, visit thermoscientific.com/pierce *N N 131 * N O H * O Thermo Scientific Amine-reactive Tandem Mass Tag Reagents Amine-reactive, sixplex and 10-plex isobaric tag reagents.

The Thermo Scientific™ Tandem Mass Tag™ Reagents are designed to enable identification and quantitation of proteins in different samples using tandem mass spectrometry (MS). Thermo Scientific™ TMT10plex™ Label Reagents share an identical structure with Thermo Scientific™ TMTzero™, TMTduplex™ and TMTsixplex™ Reagents but contain different numbers and combinations of 13C and 15N isotopes in the mass reporter. The different isotopes result in a 10-plex set of tags that have mass differences in the reporter that can be detected using high- resolution Thermo Scientific Orbitrap Mass Spectrometers. Highlights: • Powerful – concurrent MS analysis of multiple samples increases sample throughput and enables relative quantitation of up to 10 different samples derived from cells, tissues or biological fluids • Consistent – identical reagent structure and performance among TMTzero, TMTduplex, TMTsixplex and TMT10plex Reagents allow efficient transition Applications: from method development to multiplex quantitation • Protein identification and quantitation from multiple samples of cells, tissue • Robust – increased multiplex capability results in fewer missing or biological fluids quantitative values • Protein expression profiling of normal vs. disease states or • Efficient – amine-reactive, NHS-ester-activated reagents ensure efficient control vs. treated labeling of all peptides regardless of protein sequence or proteolytic • Multiplex up to 10 different samples concurrently in a single experiment enzyme specificity • Quantitative analysis of proteins for which no antibodies are available • Compatible – optimized for use with high-resolution MS/MS platforms, • Identification and quantitation of membrane and post-translationally such as Orbitrap Fusion Tribrid, Thermo Scientific™ Orbitrap Velos™ Pro, modified proteins Orbitrap Elite and Q Exactive Instruments with data analysis fully supported • Identification and quantification of hundreds to thousands of proteins in a by Proteome Discoverer 1.4 Software single experiment Advantages of the TMT10plex Reagents compared to the first-generation TMTsixplex Reagents include increased multiplex relative quantitation, increased A. TMT Reagent Generic Chemical Structure sample throughput and fewer missing quantitative values among samples. HCD O TMT10plex Reagents are ideal for the analysis of multiple protein samples O O N N from inhibitor dose response experiments, time course experiments or N O H biological replicates. ETD O

The TMT10plex Reagent Set contains 10 different isobaric compounds with Mass Mass Amine-Reactive Reporter Normalizer Group the same mass and chemical structure (i.e., isotopomeric) composed of an amine-reactive NHS-ester group, a spacer arm and a mass reporter. The reagent Figure 1. Functional regions of the Thermo Scientific TMT Reagent chemical structure set enables up to 10 different peptide samples prepared from cells or tissues to including MS/MS fragmentation sites by higher energy collision dissociation (HCD) and electron transfer dissociation (ETD). be labeled in parallel and then combined for analysis. For each sample, a unique reporter mass (i.e., TMT10 Reagent that is 126-131Da) in the low-mass region 10 6 of the high-resolution MS/MS spectrumA. TMT10plex is used to Reagents measure (TMT relative) protein B. TMTsixplex Reagents (TMT ) O O * O expression levels during peptide fragmentation and tandem mass spectrometry.O O O O O O 126 N N N N *N N * N* * * * O * N* * * * O * N * O H 126 H 129 H O O * O O O O * O O O O O O O * O O 127N *N N N * N 127C *N N N * N * N * * * O * N * * * O * N * * * O * N O H H 127 H 130 H O O O * O O * O * O * O O O O O O O * O O 128N *N * * N N * N 128C N N *N N N * * O * N * O N* O * N O H H 128 * * 131 O O H H To order, call 800.874.3723 or 815.968.0747.* Outside the U.S., contact your local* branch office or distributor.O 9 * O * O * O O O O O *N N N * N 129N * N * O * * N O 129C H H * O * O * O * O O O * O O 130N *N N N * N * * N O * N O 130C H H * O * O * O * O O *N N 131 * N O H * O A. TMT Reagent Generic Chemical Structure

HCD O O O N N N O H ETD O

Mass Mass Amine-Reactive discovery quantitationReporter Normalizer Group

Measuring changes in global protein expression

A. TMT10plex Reagents (TMT10) B. TMTsixplex Reagents (TMT6) O O * O O O O O O O 126 N N N N *N N * N* * * * O * N* * * * O * N * O H 126 H 129 H O O * O O O O * O O O O O O O * O O 127N *N N N * N 127C *N N N * N * N * * * O * N * * * O * N * * * O * N O H H 127 H 130 H O O O * O O * O * O * O O O O O O O * O O 128N *N * * N N * N 128C N N *N N N * * O * N * O N* O * N O H H 128 * * 131 O O H H * * O * O * O * O O O O O *N N N * N 129N * N * O * * N O 129C H H * O * O * O * O O O * O O 130N *N N N * N * * N O * N O 130C H H * O * O * O * O O *N N 131 * N O H * O

Figure 2. Thermo Scientific TMT Reagent chemical structures. A. TMT10plex Reagent structures with 13C and 15N heavy isotope positions (red asterisks). B. TMTsixplex Reagent structures with 13C and 15N heavy isotope positions (red asterisks).

LabelLabelLabel Label SampleSampleSample 11 1 Sample 1 TMTTMTTMT1010-126-12610-126 TMT10-126 TTreatreatTreat Denature,Denature,TreatDenature, Denature, SamplesSamplesSamples SamplesReduce,Reduce,Reduce, Reduce, SampleSampleSample 22 2 Sample 2 TMTTMTTMT1010-127N-127N10-127NTMT10-127N && IsolateIsolate& Isolate AlkylateAlkylate& IsolateAlkylate && & Alkylate & SampleSampleSample 33 3 Sample 3ProteinsProteinsProteins ProteinsTTrypticrypticTryptic TMTMTrypticTTTM1010-127C-127CT10-127CTMT10CombineCombine-127CCombine CombineFractionate,Fractionate,Fractionate, Fractionate, DigestDigestDigest Digest Clean-up,Clean-up,Clean-up, Clean-up, SampleSampleSample 44 4 Sample 4 TMTMTTTM1010-128N-128NT10-128NTMT10-128N PerformPerformPerform Perform LC-MS/MSLC-MS/MSLC-MS/MS LC-MS/MS SampleSampleSample 55 5 Sample 5 TMTMTTTM1010-128C-128CT10-128CTMT10-128C AnalysiAnalysiAnalysiss s Analysis

SampleSampleSample 66 6 Sample 6 TMTMTTTM1010-129N-129NT10-129NTMT10-129N

SampleSampleSample 77 7 Sample 7 TMTMTTTM1010-129C-129CT10-129CTMT10-129C

SampleSampleSample 88 8 Sample 8 TMTMTTTM1010-130N-130NT10-130NTMT10-130N

SampleSampleSample 99 9 Sample 9 TMTMTTTM1010-130C-130CT10-130CTMT10-130C

SampleSampleSample 1010 10 Sample 10 TMTTMTTMT1010-131-13110-131 TMT10-131

631.89 761.41 100 MS Selection MS/MS Quantitation 95 90 85

80 473.82 100 75 90 70 65 80

60 637.86 70 55 NC 60 NC 50 525.64 595.38 Figure 3. Procedure summary for MS experiments with Thermo Scientific 50 45 40 40 TMT10plex Isobaric Mass Tagging Reagents. Protein extracts isolated from cells or 466.83 35 30 NC NC 30 546.84 tissues are reduced, alkylated and digested overnight. Samples are labeled with the TMT 20 25 10 20 445.27 735.93 875.46 Reagents and then mixed before sample fractionation and clean up. Labeled samples 771.40 15 645.39 0 126 127 128 129 130 131 10 866.69 933.49 are analyzed by high-resolution Orbitrap LC-MS/MS before data analysis to identify 432.28 701.86 5 817.95 1010.81 1058.50 1168.50 1212.97 1396.78 1446.86 1542.31 m/z 0 peptides and quantify reporter ion relative abundance. 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 m/z

10 For more information, or to download product instructions, visit thermoscientific.com/pierce 131.1381 126.1277 100 95 90 127.1246 130.1409 85 80 127.1309 75 130.1348 70 e

c 65 n

a 60 128.1281 129.1376 d

n 55 u

b 50 A

e 45 v

i 129.1317 t 40 a

l 128.1341

e 35 R 30 Figure 4. Example of 10-plex relative quantitation using Thermo Scientific 10 25 TMT10plex Reagents. BSA tryptic digests labeled with TMT10plex Reagents (TMT 20 Reagent that is 126-131Da) were mixed 16:8:4:2:1:1:2:4:8:16 and analyzed by 15 high-resolution Orbitrap LC-MS. The relative abundance of the target protein or peptide 10 fragment in 10 different samples is easily measured by comparing the reporter ions 5 0 generated by MS/MS fragmentation of the different mass tags. 125.5 126.0 126.5 127.0 127.5 128.0 128.5 129.0 129.5 130.0 130.5 131.0 131.5 m/z

A. 40000 Unlabeled TMT8 35000 TMT6 TMT10 30000 No decrease 25000 in #PSMs 20000

Peptides 15000 10000 5000 0 Total Quantified Total Quantified Total Quantified Q Exactive MS Orbitrap Elite MS Orbitrap Fusion MS B. 4500 Unlabeled TMT8 4000 TMT6 TMT10 3500 3000 2500 2000

Protein Groups 1500 Figure 5. Benchmarking Thermo Scientific Orbitrap MS instruments using Thermo Scientific TMT Reagents with higher multiplexing. Number of total peptide 1000 identifications(A) and protein groups (B) are shown at 1% FDR for 500ng of Thermo 500 Scientific™ Pierce™ HeLa Protein Digest. The number of quantifiable proteins and peptides 0 is also shown. Results represent an average of two replicate runs for each sample. Total Quantified Total Quantified Total Quantified Q Exactive MS Orbitrap Elite MS Orbitrap Fusion MS

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 11 discovery quantitation

Measuring changes in global protein expression

Ordering Information

Product # Description Pkg. Size Product # Description Pkg. Size 90110 TMT10plex Labeling Reagent Set, 1 x 0.8mg 10-rxn set 90064 TMTsixplex Isobaric Mass Tagging Kit 35-rxn set Sufficient for one 10plex experiment Sufficient for five complete sixplex (5 x 6-way) experiments TMT10–126™ Label Reagent 1 x 0.8mg vial Kit Contents: TMT10–127N™ Label Reagent 1 x 0.8mg vial TMT0 Label Reagent 5 x 0.8mg vial TMT10–127C™ Label Reagent 1 x 0.8mg vial TMT6-126 Label Reagent 5 x 0.8mg vial TMT10–128N™ Label Reagent 1 x 0.8mg vial TMT6-127 Label Reagent 5 x 0.8mg vial TMT10–128C™ Label Reagent 1 x 0.8mg vial TMT6-128 Label Reagent 5 x 0.8mg vial TMT10–129N™ Label Reagent 1 x 0.8mg vial TMT6-129 Label Reagent 5 x 0.8mg vial TMT10–129C™ Label Reagent 1 x 0.8mg vial TMT6-130 Label Reagent 5 x 0.8mg vial TMT10–130N™ Label Reagent 1 x 0.8mg vial TMT6-131 Label Reagent 5 x 0.8mg vial TMT10–130C™ Label Reagent 1 x 0.8mg vial Dissolution Buffer (1M triethyl ammonium bicarbonate) 5mL TMT10–131™ Label Reagent 1 x 0.8mg vial Denaturing Reagent (10% SDS) 1mL 90111 TMT10plex Labeling Reagent Set, 3 x 0.8mg 30-rxn set Reducing Reagent (0.5 M TCEP) 1mL Iodoacetamide 12 x 9mg Sufficient for three 10plex experiments Quenching Reagent (50% hydroxylamine) 1mL TMT10–126 Label Reagent 3 x 0.8mg vial Trypsin 5 x 20μg TMT10–127N Label Reagent 3 x 0.8mg vial Trypsin Storage Solution 250μL TMT10–127C Label Reagent 3 x 0.8mg vial Albumin, Bovine 2.5mg TMT10–128N Label Reagent 3 x 0.8mg vial TMT10–128C Label Reagent 3 x 0.8mg vial 90064B TMTsixplex Isobaric Mass Tagging Kit Custom TMT10–129N Label Reagent 3 x 0.8mg vial Same formulation and kit contents as Product # 90064. TMT10–129C Label Reagent 3 x 0.8mg vial Available in custom sizes. 10 TMT –130N Label Reagent 3 x 0.8mg vial 90066 TMTsixplex Label Reagent Set, 5 x 0.8mg 30-rxn set TMT10–130C Label Reagent 3 x 0.8mg vial Sufficient for five sixplex (5 x 6-way) experiments TMT10–131 Label Reagent 3 x 0.8mg vial TMT6-126 Label Reagent 5 x 0.8mg vial 90113 TMT10plex Isobaric Mass Tag Kit 30-rxn kit TMT6-127 Label Reagent 5 x 0.8mg vial Sufficient for three 10plex experiments TMT6-128 Label Reagent 5 x 0.8mg vial Kit Contents: TMT6-129 Label Reagent 5 x 0.8mg vial TMT10–126 Label Reagent 3 x 0.8mg vial TMT6-130 Label Reagent 5 x 0.8mg vial TMT10–127N Label Reagent 3 x 0.8mg vial TMT6-131 Label Reagent 5 x 0.8mg vial 10 TMT –127C Label Reagent 3 x 0.8mg vial 90068 TMTsixplex Label Reagent Set, 2 x 5mg 72-rxn set TMT10–128N Label Reagent 3 x 0.8mg vial Sufficient for 12 sixplex (12 x 6-way) experiments TMT10–128C Label Reagent 3 x 0.8mg vial TMT6-126 Label Reagent 2 x 5mg vial TMT10–129N Label Reagent 3 x 0.8mg vial TMT6-127 Label Reagent 2 x 5mg vial TMT10–129C Label Reagent 3 x 0.8mg vial TMT6-128 Label Reagent 2 x 5mg vial TMT10–130N Label Reagent 3 x 0.8mg vial TMT6-129 Label Reagent 2 x 5mg vial TMT10–130C Label Reagent 3 x 0.8mg vial TMT6-130 Label Reagent 2 x 5mg vial TMT10–131 Label Reagent 3 x 0.8mg vial TMT6-131 Label Reagent 2 x 5mg vial Dissolution Buffer 5mL Denaturing Reagent 1mL 90063 TMTduplex Isobaric Mass Tagging Kit 15-rxn kit Reducing Reagent 1mL Sufficient for five complete duplex (5 x 2-way) experiments Iodoacetamide 12 vials x 9mg Kit Contents: Quenching Reagent 1mL TMT0 Label Reagent 5 x 0.8mg vial Pierce™ Trypsin Protease MS-Grade 5 x 20μg TMT2-126 Label Reagent 5 x 0.8mg vial Albumin, Bovine 2.5mg TMT2-127 Label Reagent 5 x 0.8mg vial Trypsin Storage Solution 250μL Dissolution Buffer (1M triethyl ammonium bicarbonate) 5mL 90406 TMT10plex Isobaric Reagent Label 60-rxn set Denaturing Reagent (10% SDS) 1mL Reagent Set Reducing Reagent (0.5M TCEP) 1mL Iodoacetamide 12 x 9mg Sufficient for 60 samples Quenching Reagent (50% hydroxylamine) 1mL TMT10–126 Label Reagent 1 x 5mg vial Trypsin 2 x 20μg TMT10–127N Label Reagent 1 x 5mg vial Trypsin Storage Solution 250μL TMT10–127C Label Reagent 1 x 5mg vial Albumin, Bovine 2.5mg TMT10–128N Label Reagent 1 x 5mg vial TMT10–128C Label Reagent 1 x 5mg vial 90065 TMTduplex Isobaric Label Reagent Set, 5 x 0.8mg 10-rxn set TMT10–129N Label Reagent 1 x 5mg vial Sufficient for five duplex (5 x 2-way) experiments TMT10–129C Label Reagent 1 x 5mg vial TMT2-126 Label Reagent 5 x 0.8mg TMT10–130N Label Reagent 1 x 5mg vial TMT2-127 Label Reagent 5 x 0.8mg 10 TMT –130C Label Reagent 1 x 5mg vial 90060 TMTduplex Isotopic Label Reagent Set, 5 x 0.8mg 10-rxn set TMT10–131 Label Reagent 1 x 5mg vial Sufficient for five duplex (5 x 2-way) experiments 90061 TMTsixplex Label Reagent Set, 1 x 0.8mg 6-rxn set TMT0 Label Reagent 5 x 0.8mg Sufficient for one sixplex (1 x 6-way) experiments TMT6-127 Label Reagent 5 x 0.8mg 6 TMT -126 Label Reagent 1 x 0.8mg vial 90067 TMTzero Label Reagent, 5 x 0.8mg 5-rxn set TMT6-127 Label Reagent 1 x 0.8mg vial Sufficient for controls in five experiments TMT6-128 Label Reagent 1 x 0.8mg vial TMT0 Label Reagent, 5 x 0.8mg TMT6-129 Label Reagent 1 x 0.8mg vial TMT6-130 Label Reagent 1 x 0.8mg vial 90114 1M Triethylammonium Bicarbonate (TEAB) 50mL 6 TMT -131 Label Reagent 1 x 0.8mg vial 90115 50% Hydroxylamine 5mL 90062 TMTsixplex Label Reagent Set, 2 x 0.8mg 12-rxn set Sufficient for two sixplex (2 x 6-way) experiments TMT6-126 Label Reagent 2 x 0.8mg vial TMT6-127 Label Reagent 2 x 0.8mg vial For a list of references using amine-reactive TMT Reagents, TMT6-128 Label Reagent 2 x 0.8mg vial please see the back cover. TMT6-129 Label Reagent 2 x 0.8mg vial TMT6-130 Label Reagent 2 x 0.8mg vial TMT6-131 Label Reagent 2 x 0.8mg vial

12 For more information, or to download product instructions, visit thermoscientific.com/pierce Thermo Scientific Cysteine-reactive Tandem Mass Tag Reagents For protein expression analysis of sulfhydryl groups by mass spectrometry.

Thermo Scientific Tandem Mass Tag (TMT) Reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry. The Thermo Scientific™ iodoTMT™ Reagents are sets of isobaric isomers (i.e., same mass and structure) that contain iodoacetyl function groups for covalent, irreversible labeling of sulfhydryl (–SH) groups. Similar to iodoacetamide, iodoTMT Reagents react specifically with reduced cysteines in peptides and proteins. iodoTMT Reagents can be differentiated by tandem mass spectrometry (MS/MS), enabling identification and relative quantitation of cysteine modifications, such as S-nitrosylation, oxidation and disulfide bonds, across different samples or experimental conditions. IodoTMT Reagents replace our previously offered Thermo Scientific™ CysTMT™ Reagents, which utilized a dithiopyridine reactive group to reversibly label cysteine sulfhydryls. Highlights: Applications: • Specific – only reacts with reduced sulfhydryl groups • Identify and quantify low abundant cysteine-containing peptide subproteome • Irreversible – labeled proteins and peptides are not susceptible • Combine up to six different samples or experimental conditions in a single to reducing agents LC-MS analysis • Flexible – options for duplex isotopic (MS) or sixplex isobaric • Determine sites of cysteine post-translational modifications (MS/MS) quantitation (e.g., S-nitrosylation, oxidation and disulfide bonds) • Complete – workflow combines efficient labeling, enrichment and elution • Measure cysteine post-translational modification occupancy across different for identification and quantitation of cysteine-containing peptides samples or experimental conditions

A. Thermo Scientific iodoTMTzero Reagent Conjugation Reaction CID O O H Cysteine R’ N N N N I 126Da H H NH ETD O HS + O Mass Mass Cys-Reactive R Reporter Normalizer Group

R’ O O H N N NH N N S H H O R O

Figure 1. Mechanism of Thermo Scientific iodoTMT Reagent reaction with cysteine-containing proteins or peptides.

B. Thermo Scientific iodoTMTsixplex Reagents

* O O O O H H N N * N N * N* * * * N I * N * N I 126 H H 129 H H O To order, call* 800.874.3723 or 815.968.0747.O Outside the U.S., contact your local branch office or distributor. 13 * O O * O O H H * N N N * N * N * * * N I * N N I 127 H H 130 H H O * O

* * O O * O O H H N N * N N * N* * N I * N N I 128 H H 131 H H * O * O A. Thermo Scientific iodoTMTzero Reagent Conjugation Reaction CID O O H Cysteine R’ N N N N I 126Da H H NH ETD O HS + O Mass Mass Cys-Reactive R Reporter Normalizer Group

R’ O O H N N NH N N S H H discovery quantitationO R O Measuring changes in global protein expression

B. Thermo Scientific iodoTMTsixplex Reagents

* O O O O H H N N * N N * N* * * * N I * N * N I 126 H H 129 H H O * O

* O O * O O H H * N N N * N * N * * * N I * N N I 127 H H 130 H H O * O

* * O O * O O H H N N * N N * N* * N I * N N I 128 H H 131 H H * O * O

Figure 2. Structure of Thermo Scientific iodoTMTsixplex Reagents for cysteine labeling, enrichment and isobaric MS quantitation.

Samples Label 1 iodoTMT6-126 Combine; Enrich for Treat Remove TMT-tagged excess peptide using 2 samples; iodoTMT6-127 Isolate Denature; reagent; anti-TMT proteins 6 Digest resin 3 Reduce iodoTMT -128

4 iodoTMT6-129

iodoTMT6-130 5 = TMT Peptide Label (or protein) 6 6 Sample 1 TMT10-126 iodoTMT -131 Treat Denature, Samples Reduce, Sample 2 TMT10-127N & Isolate Alkylate & Sample 3 Proteins Tryptic TMT10-127C Combine Fractionate, Digest Clean-up, Fractionate; Sample 4 10 Perform TMT -128NClean-up; LC-MS/MS LC-MS/MS Sample 5 TMT10-128Canalysis Analysis Sample 6 TMT10-129N

Sample 7 TMT10-129C

Sample 8 TMT10-130N

Sample 9 TMT10-130C

Sample 10 TMT10-131

Figure 3. Schematic of Thermo Scientific iodoTMTsixplex Reagent workflow. Six different sample conditions can be prepared for iodoTMT Reagent labeling. Labeled proteins are combined before iodoTMT-labeled peptide enrichment using immobilized anti-TMT antibody resin and subsequent LC-MS/MS analysis of isobaric reporter ions.

14 For more information, or to download product instructions, visit thermoscientific.com/pierce A. Specificity of Enrichment B. Distribution of Enrichment C. Composition of Unique Proteins

100 5000 Unenriched 90 4500 Enriched 80 4000

70 3500 Unenriched Enriched 60 3000

50 2500

40 2000 438 552 282 30 2500

20 1000 Nunmber of Peptides or Proteins Nunmber of Peptides % Total of Peptides ( = Labeled) of Peptides Total % 10 500

0 0 Not Acid TMT Unique Unique Enriched Elution Elution Peptides Proteins Buffer Buffer Enriched

Figure 4. Anti-TMT enrichment of iodoTMT-labeled peptides. Panel A: Percent of iodoTMT-labeled peptide modifications from A549 cell lysate proteins identified before and after enrichment using acidic and TMT elution buffers. Panel B: Comparison of total number of unique peptides and proteins identified before and after anti-TMT antibody resin enrichment. Panel C: Venn diagram of unique proteins identified before and after anti-TMT antibody resin enrichment.

8000 SNOC SNOC 7000 2500 6000 G c I T I I G G G D T A T c c A K LPS 5000 LPS

y₁₂²⁺ 2000 741.86 4000 (-)

3000 (-) Intensity (counts)

2000 1500 1000

0 126 127 128 129 130 131 1000 y₁₀²⁺ y₁₄²⁺ iodoTMTsixplex channel Intensity (counts) 656.84 848.94

b₁₆²⁺ b₁₄²⁺ 940.99 y₇²⁺ 825.63 y₈²⁺ b₁₅²⁺, y₁₅²⁺ 500 542.51 y₁₁²⁺ 599.70 y₁₃²⁺ 905.48 y₅²⁺ 685.51 798.48 b₁₁⁺ 456.27 b₅⁺ y₉²⁺ y₃⁺ 1045.61 b₃⁺ 545.22 628.26 b₂⁺, y₂⁺ 378.28 y₁₆²⁺ b₁₂⁺ 331.09 218.20 985.85 1116.70 0 200 400 600 800 1000 1200 m/z

Figure 5. Measured induction of S-nitrosylation in phosphoglycerate kinase 1 peptide. BV-2 glioma cells were either untreated or treated with lipopolysaccharide (LPS) or S-nitrosocysteine (SNOC) for 20 hours to induce S-nitrosylation and selectively labeled with iodoTMTsixplex Reagents using the S-nitrosylation switch assay. MS spectrum includes phosphoglycerate kinase 1 peptide (GcITIIGGGDTATccAK, inset) showing localization of the iodoTMT-modified cysteine (red). Inserted graph shows an increase in S-nitrosylation of phosphoglycerate kinase 1 peptide in response to S-nitrosylation inducing agents lipopolysaccharide (LPS, 127 & 130) and S-nitrosocysteine (SNOC, 129 &131) determined by relative quantitation of TMT reporter ions in duplicate samples.

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 15 discovery quantitation

Measuring changes in global protein expression

Ordering Information

Product # Description Pkg. Size 90100 iodoTMTzero Label Reagent Set, 5 x 0.2mg 5-rxn set Sufficient for controls in five experiments iodoTMT0-126 Label Reagent 5 x 0.2mg 90101 iodoTMTsixplex Label Reagent Set, 1 x 0.2mg 6-rxn set Sufficient for one sixplex (1 x 6-way) experiment iodoTMT6-126 Label Reagent 1 x 0.2mg iodoTMT6-127 Label Reagent 1 x 0.2mg iodoTMT6-128 Label Reagent 1 x 0.2mg iodoTMT6-129 Label Reagent 1 x 0.2mg iodoTMT6-130 Label Reagent 1 x 0.2mg iodoTMT6-131 Label Reagent 1 x 0.2mg 90102 iodoTMTsixplex Label Reagent Set, 5 x 0.2mg 30-rxn set Sufficient for five sixplex (5 x 6-way) experiments iodoTMT6-126 Label Reagent 5 x 0.2mg iodoTMT6-127 Label Reagent 5 x 0.2mg iodoTMT6-128 Label Reagent 5 x 0.2mg iodoTMT6-129 Label Reagent 5 x 0.2mg iodoTMT6-130 Label Reagent 5 x 0.2mg iodoTMT6-131 Label Reagent 5 x 0.2mg 90103 iodoTMTsixplex Isobaric Mass Tag Labeling Kit 30-rxn kit Sufficient for five complete sixplex (5 x 6-way) experiments Kit Contents: iodoTMT6-126 Label Reagent 5 x 0.2mg iodoTMT6-127 Label Reagent 5 x 0.2mg iodoTMT6-128 Label Reagent 5 x 0.2mg iodoTMT6-129 Label Reagent 5 x 0.2mg iodoTMT6-130 Label Reagent 5 x 0.2mg iodoTMT6-131 Label Reagent 5 x 0.2mg HES Buffer 15mL Bond-Breaker™ TCEP Solution 0.5mL DTT, No-Weigh™ Format 8 x 7.7mg Trypsin 5 x 20μg Trypsin Storage Solution 250μL Albumin (bovine) 2.5mg 90075 Anti-TMT Antibody 0.1mL Sufficient for detection of the TMT label in Western blots 90076 Immobilized Anti-TMT Antibody Resin 6mL Sufficient for binding 250μg TMT-BSA/mL resin 90104 TMT Elution Buffer 20mL Sufficient for elution of TMT-labeled peptides from immobilized anti-TMT antibody resin 90106 HENS Buffer 100mL Sufficient for detection of the TMT label in Western blots

16 For more information, or to download product instructions, visit thermoscientific.com/pierce Thermo Scientific Carbonyl-reactive Tandem Mass Tag Reagents Enable characterization and multiplex quantitation of carbonyl-containing biomolecules.

The Thermo Scientific™ aminoxyTMT™ Mass Tag Labeling Reagents enable multiplexed relative quantitation of carbonyl-containing compounds by mass spectrometry (MS). The six compounds of the Thermo Scientific™ aminoxyTMTsixplex™ Reagent Set have the same nominal mass (i.e., isobaric) and chemical structure (carbonyl-reactive aminoxy group, spacer arm and mass reporter). However, the specific distribution of13 C and 15N isotopes on either side of the high energy collision dissociation (HCD) or electron transfer dissociation (ETD) MS/MS fragmentation site in each reagent results in a unique reporter mass (126-131Da) in the low mass A. aminoxyTMTzero Reagent region. This distribution of reporter masses is used to measure the relative abundance HCD of labeled molecules in a combined (multiplexed) MS sample representing six O O N NH2 N N O different treatment conditions. The aminoxyTMT Reagents may be used to quantify 126Da H H ETD a broad range of biologically important molecules including carbohydrates, steroids or oxidized proteins. Mass Mass Carbonyl-Reactive Applications: Reporter Normalizer Group • Relative quantitation of glycans For glycobiology MS applications, native glycans are difficult to study by mass • Study of structural diversity of protein glycosylation spectrometry because of their poor ionization efficiency. Quantitation of glycans aminoxyTMTsixplex Reagents • Study of glycosylation in cell signaling and regulation is particularly challenging due to the lack of standards for all naturally occurring * • Study of cancerO progression,O biomarker discovery and analysisO of biotherapeuticsO glycans and difficulties reproducibly quantifying multiple samples. The aminoxy N NH2 * N NH2 * N* * * * N O * N * N O group has better reactivity with carbonyls and better stability of the labeled • 126Study of proteinH oxidationH 129 H H * product compared to hydrazide groups. Labeling with the aminoxyTMT Reagents * improves ionization of glycans, thus improving sensitivity, and enables relative A. aminoxyTMTzeroO ReagentO * O O *N HCD NH2 N * NH2 quantitation of glycans for up to six samples concurrently. * N * * * N O O O * N N O 127 H H 130 H H N NH2 N N O * 126Da H H Highlights: * ETD * O O * O O • Quantitative – enables relative quantitation of glycans or other carbonyl- Mass Mass Carbonyl-Reactive N * NH2 *N NH2 * N * ReporterN NormalizerO Group* N N O reactive proteins from multiple samples of cells, tissues or biological fluids 128 H H 131 H H • Stable – stable product formed after labeling reaction * * B. Labeling the reducing end carbonyl of a glycan with aminoxyTMT Reagent • Efficient – achieve labeling efficiency greater than 90% in one hour aminoxyTMTsixplex Reagents OH OH • Sensitive – labeled glycan signal-to-noise ratio improved greater than 20-fold * RO O O O RO OH O O when compared to native glycan OH O HO N HO NH2 * N NH2 * N* * * * N O * N * N O • Multiplex – identify and characterize up to six samples concurrently 126 NHAc H H NHAc 129 H H * • Optimized – procedure and reagents optimized for excellent labeling aminoxyTMT H O * O O *2 O O efficiency and recovery of glycans Reagent *N NH2 N NH2 * N * * * N O * N* N O 127 OH 130 H H O H O H RO OH * HO N N * O N * N O O NHAc *H O H O N NH Oxime Bond*N NH * N* * N O 2 * N N O 2 128 H H 131 H H Figure* 1. Thermo Scientific aminoxyTMT Reagent chemical* structures. A. Functional regionsB. Labeling of the the aminoxyTMTzero reducing end carbonyl Reagent ofstructure a glycan including with aminoxyTMT MS/MS fragmentation Reagent sites by HCD and ETD. B. Reaction scheme for labeling of reducing-end sugars with aminoxyTMT Reagent. OH OH

RO O RO OH HO OH HO O NHAc NHAc

aminoxyTMT H2O Reagent OH O O RO OH N N HO O N N NHAc H H Oxime Bond To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 17 A. aminoxyTMTzero Reagent HCD discoveryO O quantitation N NH2 N N O 126Da H H ETD

MeasuringMass changesMass in globalCarbonyl-Reactive protein expression Reporter Normalizer Group

aminoxyTMTsixplex Reagents * O O O O N NH2 * N NH2 * N* * * * N O * N * N O 126 H H 129 H H * * O O * O O *N NH2 N NH2 * N * * * N O * N* N O 127 H H 130 H H * * * O O * O O N NH *N NH * N* * N O 2 * N N O 2 128 H H 131 H H * * B. Labeling the reducing end carbonyl of a glycan with aminoxyTMT Reagent Figure 2. Thermo Scientific aminoxyTMTsixplex Reagent structures and isotope positions (*). OH OH

RO O RO OH HO OH HO O NHAc NHAc

aminoxyTMT H2O Reagent OH O O mAb1 – 126, 129 RO OH N N HO O N N

H H 3_mAbs_123123_894 #10 RT: 0.10 AV: 1 NL: 5.69E3 NHAc T: ITMS + p ESI Full ms2 [email protected] [50.00-1500.00] 126.25 131.34 100 128.34 130.34 mAb2 – 127, 130 95

90 127.34 N -glycans N -glycans N -glycans 129.34 Oxime Bond 85

80

75

70

65

60

55 mAb3 – 128, 131

50 bundan ce A

e v i t 45 a l e R 40

35 126 127 130 128 131 HCD30 129 TMT TMT TMT 25 TMT TMT TMT 20

15 124.25 10

5 125.25 3_mabs_123123_975 #21 132RT.2: 50.24 AV: 1 NL: 5.48E3 122.25 123.25 T: ITMS + p ESI Full ms2 [email protected] [50133.00.-215005 .00]134.25 135.17 0 126.25 122 123 124 125 126 127 128 129 130 100 131 132 133 134 135 m/z 95 129.34 90 3_mAbs_123123 #54 RT: 0.11 AV: 1 NL: 2.41E6 G0F Reporter ions85 T: ITMS + p ESI Full ms [700.00-1500.00] 80 893.84 75 100 70

65

95 60

55

50 90 bundan ce A

e v i

MS t 45 a l e 85 R 40 35 127.34 128.34 130.34 HCD 131.34 80 30 Combined sample 25 75 20 15

10

70 5 124.17 125.25 132.25 122.25 123.25 133.25 134.25 135.17 0 122 123 124 125 126 127 128 129 130 131 3_mabs13_123123_1052 6133 #8 RT: 130.410 AV: 113N5L: 1.22E3136 65 m/z T: ITMS + p ESI Full ms2 [email protected] [50.00-1500.00] 126.25 G1F 100 60 95 974.92 Reporter ions90 55 85 129.34 80 50 75 bundan ce

A 70

v e 65 i t 45 a

l 60 e R 40 55 50 bundan ce

HCD A

e v i t 45 a l e

35 R 40

35 30 30

25

25 20 127.25 128.34 130.34 15 131.34

20 10

5 G2F 124.17 125.25 122.25 123.25 132.25 15 882.84 901.84 0 122 123 124 125 126 127 128 129 130 131 132 133 1055.93 m/z 10 904.84 963.92 982.84 820.84 873.34 5 Reporter ions 792.34 829.75 910.76 954.34 991.84 1040.18 1063.84 1140.10 855.67 1098.09 1168.18 1198.10 1226.01 0 800 850 900 950 1000 1050 1100 1150 1200 m/z

Figure 3. Relative quantitation of glycans from monoclonal antibodies. N-glycans were released from three different mouse monoclonal antibodies (approximately 100μg each) using PNGase F glycosidase. Each of the three glycan samples was split into two equal parts and labeled with a different mass tag, indicated in the figure. After labeling, quenching and clean-up, the samples were mixed and analyzed by direct infusion ESI-MS in the positive ion mode on a Velos Pro Mass Spectrometer. Glycoforms of interest were identified in the MS spectra and were subjected to HCD MS/MS. Reporter ion relative peak intensities provide information on relative glycoform abundance in the three samples (insets).

18 For more information, or to download product instructions, visit thermoscientific.com/pierce A. Glc7_100µgmL_unlabeled # 35 RT: 0.11 AV: 1 NL: 1.91E7 T: ITMS + p ESI Full ms [50.00–25000.00]

599.43 100 Glc7 (unlabeled) 95 [M+2Na]2+ 90 Glc7 (unlabeled) Native unlabeled Glc7 85 + 80 [M+Na] 100µg/mL 75 1175.69 70 ESI solution: 50% ACN, 50µM NaOH 65 60 Velos Pro Mass Spectrometer 55 bundan ce 50 A 45 v e i

t 40 a l 35 e

R 30 25 20 15 10 736.60 437.43 5 494.84 791.43 851.52 1013.61 1225.11 413.51 648.43 926.27 1095.11 1331.03 1449.95 1560.37 1642.12 1752.87 1868.29 1945.21 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

B. Glc7_2µgmL_oxyTMT # 35 RT: 0.11 AV: 1 NL: 2.21E7 T: ITMS + p ESI Full ms [50.00–20000.00]

736.60 100 Glc7 (labeled) aminoxyTMT Reagent-labeled Glc7 95 2+ 90 [M+H+Na] 2µg/mL 85 20- to 50-fold increase in S/N 80 75 70 65 60 55 No residual reagent

bundan ce 50 A 45 v e i

t 40 a l 35 e

R 30 Glc7 (labeled) 25 [M+H]+ 20 15 747.52 10 5 360.59 502.09 655.51 574.51 830.68 974.60 1068.19 1129.36 1270.28 1353.03 1449.87 1513.53 1699.37 1787.54 1896.04 1955.79 0 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 m/z

Figure 4. Positive ion mode electrospray ionization (ESI) of native unlabeled and aminoxyTMT-labeled maltoheptaose (Glc7) standard. Panel A: MS spectrum of native unlabeled Glc7; Panel B: MS spectrum of aminoxyTMT-labeled Glc7 standard. Fifty-fold lower concentration of the aminoxyTMT-labeled sample solution produced similar base peak intensity as the unlabeled sample. Residual aminoxyTMT reagent or unlabeled Glc7 were not detected Ordering Information after labeling and sample clean-up (Panel B).

Product # Description Pkg. Size 90400 aminoxyTMTzero Label Reagent Set, 6-rxn set 6 x 0.2mg Sufficient for controls in six experiments 6 x 0.2mg vial aminoxyTMT0 Label Reagent 90401 aminoxyTMTsixplex Label Reagent Set, 6-rxn set 1 x 0.2mg Sufficient for six reactions aminoxyTMT6–126 Label Reagent 1 x 0.2mg vial aminoxyTMT6–127 Label Reagent 1 x 0.2mg vial aminoxyTMT6–128 Label Reagent 1 x 0.2mg vial aminoxyTMT6–129 Label Reagent 1 x 0.2mg vial aminoxyTMT6–130 Label Reagent 1 x 0.2mg vial aminoxyTMT6–131 Label Reagent 1 x 0.2mg vial 90402 aminoxyTMTsixplex Label Reagent Set, 30-rxn set 5 x 0.2mg Sufficient for five sixplex reactions aminoxyTMT6–126 Label Reagent 5 x 0.2mg vial aminoxyTMT6–127 Label Reagent 5 x 0.2mg vial aminoxyTMT6–128 Label Reagent 5 x 0.2mg vial aminoxyTMT6–129 Label Reagent 5 x 0.2mg vial aminoxyTMT6–130 Label Reagent 5 x 0.2mg vial aminoxyTMT6–131 Label Reagent 5 x 0.2mg vial

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 19 targeted quantitation Measuring expression of selected proteins

Quantitative proteomic experiments are increasingly used in pharmaceutical and diagnostic applications to quantify proteins in complex samples. These methods typically minimize the amount of sample preparation to improve precision and throughput. Targeted quantitative proteomic workflows involve protein denaturation, reduction, alkylation, digestion and desalting prior to LC-MS/ MS analysis. To improve assay sensitivity and selectivity, immunoprecipitation or abundant protein depletion may be used prior to sample processing. To further improve quantitative precision and accuracy, known amounts of synthetic peptides containing heavy stable isotopes, such as Thermo Scientific™ HeavyPeptide™ Reagents, are added to samples prior to MS analysis. These peptides serve as internal quantitative standards for absolute quantification of the corresponding natural peptides in a biological sample. To better control for digestion efficiency, heavy proteins synthesized with the Thermo Scientific™ 1-Step Heavy Protein IVT Kit can be spiked into the sample prior to digestion, and then the resulting heavy peptides can be used for relative quantitation across all samples. Targeted quantitative protein studies are typically performed on triple quadrupole mass spectrometers, such as the Thermo Scientific™ TSQ Quantiva™ Triple Stage Quadrupole Mass Spectrometer. A triple quadrupole mass spectrometer Targeted proteomics experiments are typically quantifies peptides by serially monitoring specific mass windows for peptides designed to quantify less than 100 proteins with of interest, isolating the peptide(s), fragmenting and then quantifying several very high precision, sensitivity, specificity and fragment ions specific for each peptide of interest. This selective reaction monitoring (SRM) strategy for targeted quantitation, along with chromatographic throughput. Targeted MS quantitation strategies retention time information, provides high sensitivity and specificity. Alternatively, use specialized workflows and instruments to high resolution and accurate mass instruments, such as the Q Exactive Mass improve the specificity and quantification of a limited Spectrometer, are being used to quantify proteins with even greater selectivity. ™ number of features across hundreds or thousands Specialized software such as Thermo Scientific Pinpoint 1.4 Software ensures as much high-quality data is acquired, and as much valuable information is of samples. extracted from that data, as possible.

20 For more information, or to download product instructions, visit thermoscientific.com/pierce Targeted Quantitation Workflow by Sample Type Using Thermo Scientific Products

Serum/Plasma Mammalian Cells Tissue Biofluids

• Lysis & Fractionation Reagents • Abundant Protein Depletion Reagents (Mass Spec Sample Prep Kit for (Albumin, Top 2 or Top 12 Protein Cultured Cells) Depletion Spin Columns and Kits)

• Targeted Protein Digestion Standard (1-Step Heavy Protein In Vitro Protein Expression Kit)

• Protein Immunoprecipitation (Pierce IP/co-IP Kits)

• Protein Quantitation Reagents (BCA and Micro BCA Protein Assay Kits)

• Protein Digestion Verification (Digestion Indicator for Mass Spectrometry) • Protein Digestion - Proteases (Trypsin, LysC, LysN, Chymotrypsin, AspN, and/or GluC Proteases, MS Grade)

• Peptide Enrichment (Fe-NTA and/or TiO2 Phosphopeptide Enrichment Columns and Kits)

• Protein Quantitation Standards (HeavyPeptide AQUA Standards, PEPotech SRM Libraries) • Peptide Retention Time Calibration Mixture

• Peptide Clean-Up (Detergent Removal Spin Columns) • Peptide Clean-Up (C18, Graphite Spin Columns and Tips)

Instruments • TSQ Vantage Triple Quadrapole Mass Spectrometer • TSQ Endura Triple Quadrapole Mass Spectrometer • TSQ Quantiva Triple Quadrapole Mass Spectrometer • Q Exactive Mass Spectrometer

Software • Pinpoint 1.4 Software

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 21 targeted quantitation

Measuring expression of selected proteins

Thermo Scientific HeavyPeptide AQUA Standards High-quality isotopically labelled peptides for absolute quantitation.

The Thermo Scientific™ HeavyPeptide™ AQUA Custom Synthesis Service provides isotopically labelled, AQUA-grade peptides for the relative and absolute quantitation of proteins at very low concentrations in complex protein mixtures.

HeavyPeptide sequences up to 15 amino acids in length are synthesized using the latest Fmoc solid-phase technology and purified by HPLC. All HeavyPeptide sequences are analyzed by mass spectrometry, and AQUA- (Absolute QUAntitation), Ultimate- and QuantPro-grade peptides are validated using stringent analytical HPLC to determine the final purity and assure that you are receiving only the highest quality peptides for absolute quantitation. We offer advanced heavy peptide synthesis capabilities with a Includes: wide range of labels, modifications, scales and purities to meet your research • One isotopically labelled peptide or non-labelled control peptide sequence needs. HeavyPeptide AQUA-grade peptides are also part of our Thermo Applications: ™ ™ Scientific HeavyPeptide FasTrack Service to accelerate targeted assay • Biomarker discovery, verification and validation development. • Functional quantitative proteomics • Quantitation of post-translational modifications These peptides are packaged using our Thermo Scientific™ ArgonGuard service, • Confirmation of RNA interference (RNAi) in which peptides are packaged in argon gas to minimize amino acid oxidation during shipping and storage. This standard service helps maintain biological • Pharmacokinetics activity of custom peptides and reduce experimental variation. • Metabolomics • Clinical biochemistry for drug and metabolite monitoring Highlights: • Anti-doping testing • Accurate – concentration precision to meet your application needs • Protein expression monitoring • Consistent results – ArgonGuard service helps maintain biological activity of peptides • Cell signal profiling and pathway validation • Sensitive – enables the absolute quantification of low-abundant proteins • Specific – 100% peptide sequence specificity • Flexible – extensive list of available modifications, solvents, concentrations, aliquots and formats

Table 1. Thermo Scientific HeavyPeptide AQUA Grades.

Grade Description AQUA Ultimate Fully solubilized peptides with a concentration precision ±5%. The best choice for biomarker validation and experiments demanding ultimate quantitative precision and batch-to-batch reproducibility. AQUA QuantPro Fully solubilized peptides with a concentration precision ±25% that are ideal for biomarker verification. AQUA Basic Lyophilized peptides that are more adequate for relative quantitation. The batch-to-batch consistency is not quantitated.

22 For more information, or to download product instructions, visit thermoscientific.com/pierce Table 2. Specifications of Thermo Scientific HeavyPeptide AQUA-Grade Standards.

Grade AQUA Ultimate AQUA QuantPro AQUA Basic

Formulation 5pmol/μL in 5% (v/v) acetonitrile/H2O 5pmol/μL in 5% (v/v) acetonitrile/H2O Lyophilized Actual concentration Measured by AAA† Measured by AAA† Measured by AAA† Concentration precision ±5% ±25% NA Peptide purity >97% >97% >95% Isotopic enrichment >99% >99% >99% Peptide length Up to 30 amino acids Up to 30 amino acids Up to 30 amino acids Amount/No. of aliquots 10nmol/10 aliquots 10nmol/10 aliquots 15 to 30nmol†† (0.05 to 0.1mg)/aliquot 40nmol/40 aliquots 40nmol/40 aliquots 96nmol/96 aliquots 96nmol/96 aliquots Quality control MS & analytical HPLC, AAA (+/-5-10%) MS & analytical HPLC, AAA (+/-25%) MS & analytical HPLC Delivery time§ 6-8 weeks 6-8 weeks 6-8 weeks Shipment In solution on wet ice In solution on wet ice Lyophilized at room temp. Product options • Additional light amino acids to extend the peptide length • Additional heavy amino acid on each peptide • Multiple solvents, concentrations and aliquot sizes available • Peptides delivered in various formats (i.e., 96-well plate with or without detachable tubes in glass or plastic, 2D barcodes, etc.) Peptide modifications • Single or double phosphorylation (pY, pT or pS) • Cysteine carbamidomethylation (CAM)‡ • Chloro-L-Tyrosine • Pryoglutamic acid • Methionine oxidation (Met[O]) • Other modifications available on request † Amino acid analysis. § These production times are estimates that vary based on the number of peptides ordered. †† 30nmol is valid for peptides 6-15 amino acids in length. For shorter or longer peptides, the amount might decrease to as little as 15nmol. ‡ CAM tends to cause cyclisation at the N-terminus. Fully cyclized form can be provided upon request.

Table 3. Heavy amino acids offered with Thermo Scientific HeavyPeptide Custom Synthesis.†

Amino acid Code Mass difference Isotope Isotopic enrichment

13 15 Alanine A +4Da U- C3, N >99%

13 15 Arginine R +10Da U- C6, N4 >99%

13 15 Isoleucine I +7Da U- C6, N >99%

13 15 Leucine L +7Da U- C6, N >99%

13 15 Lysine K +8Da U- C6, N2 >99%

13 15 Phenylalanine F +10Da U- C9, N >99%

13 15 Proline P +6Da U- C5, N2 >99%

13 15 Valine V +6Da U- C5, N2 >99% † Other amino acids on request.

For a list of references using HeavyPeptide Reagents, please see the back cover.

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 23 targeted quantitation

Measuring expression of selected proteins

Thermo Scientific HeavyPeptide Highlights: AQUA FasTrack Service • Efficient – no need to resynthesize selected peptides for target quantitation • Accurate – concentration precision to meet your application needs Maximize your budget using two-step targeted assay • Sensitive – enables the absolute quantification of low-abundant proteins development for SRM. • Consistency – peptides from the same synthesis reaction are used for both assay development and quantitation The HeavyPeptide AQUA FasTrack Service is a two-step approach to accelerate targeted assay development and absolute quantitation by selected reaction Applications: monitoring (SRM) within a controlled-budget environment. • Biomarker discovery, verification and validation • Functional quantitative proteomics Quantitative proteomics workflows usually begin with the software-assisted • Quantitation of post-translationally modified proteins selection of proteotypic peptide candidates. After synthesis, these crude peptides are used to identify the best peptide candidates and optimize the quantitative • Confirmation of results from RNA interference (RNAi) liquid chromatography-mass spectrometry (LC-MS) assay. Highly pure • Pharmacokinetics HeavyPeptide sequences are then synthesized and purified for target quantitation. • Metabolomics Traditionally, these steps required two independent syntheses, which are • Clinical biochemistry for drug and metabolite monitoring time-consuming and expensive. The HeavyPeptide FasTrack Service combines • Anti-doping testing targeted assay development and quantitation for a rapid, precise workflow that • Protein expression monitoring saves you time and money. • Cell signal profiling and pathway validation The HeavyPeptide FasTrack Service provides highly pure AQUA-, Ultimate- or QuantPro-grade peptides through two phases:

FasTrack 1: Crude HeavyPeptide sequences (up to 96 per order) are synthesized within two weeks, and a 100μg aliquot of each peptide is shipped for proteotypic peptide FasTrack 1 FasTrack 2 selection and assay development. The remainder of each crude peptide is stored Synthesis of Synthesis of until optimization is completed. The customer then indicates the specific peptides peptide candidates peptide candidates and number of aliquots required for target quantitation. Targeted Assay Target Development Quantitation FasTrack 2 (optional): Because the crude peptides selected in FasTrack phase 1 have already been synthesized, there is no need to resynthesize the peptides for target quantitation by SRM. The selected crude peptides are purified to a minimum purity of 97% and fully solubilized using a proprietary process. The HeavyPeptide concentrations are measured by amino acid analysis, and quality control is performed using MALDI MS and analytical HPLC. AQUA-grade peptides are then shipped on wet ice for target quantitation in three weeks or less.

Another cost-effective alternative for the medium- to high-throughput development of SRM assays as part of the targeted assay development are Thermo Scientific™ PEPotec™ SRM Custom Peptide Libraries (see pgs. 26-27), which can be supplied in library sizes from 24 to >1000 peptides.

24 For more information, or to download product instructions, visit thermoscientific.com/pierce Targeted Assay Development Target Quantitation Search spectral and Analyze results Perform absolute target peptide libraries and and identify optimum FasTrack 2 quantitation with highly pure perform in silico proteotypic peptides AQUA Ultimate or predictions to identify QuantPro-grade sequences for assay peptides development

Develop target assay using crude HeavyPeptide sequences Analyze results and quantitate target peptides FasTrack 1 LC-MS

Figure 2. The Thermo Scientific HeavyPeptide FasTrack Service accelerates target assay development and quantitation. A key step for targeted quantitation is identifying sequence-specific or unique target peptides with sufficient signal intensity for the absolute quantitation of the target peptide(s). This step begins with a combination of peptide and spectral database searches and computational (in silico) predictions using programs such as Pinpoint Software to identify an initial list of potential peptides that corresponds to multiple proteins of interest. Crude HeavyPeptide sequences are synthesized in FasTrack phase 1 and screened by LC-MS. Results are analyzed to optimize LC and MS workflows and identify the best proteotypic sequences for target quantitation. The selected crude peptides are purified in FasTrack phase 2 and used for absolute target quantitation by SRM via LC-MS.

Table 1. Thermo Scientific HeavyPeptide AQUA grades.

Grade Description AQUA Ultimate Fully solubilized peptides with a concentration precision +/-5% that are the best choice for biomarker validation and experiments demanding ultimate quantitative precision and batch-to-batch reproducibility. AQUA QuantPro Fully solubilized peptides with a concentration precision ±25% that are ideal for biomarker verification.

Table 2. Specifications of Thermo Scientific HeavyPeptide† FasTrack phases.

Phase FasTrack 1 FasTrack 2 Grade Crude AQUA Ultimate AQUA QuantPro

Formulation Lyophilized 5pmol/μL in 5% (v/v) acetonitrile/H2O 5pmol/μL in 5% (v/v) acetonitrile/H2O Peptide purity Crude >97% >97% Amount/No. 0.1mg/1 aliquot 10nmol/10 aliquots 10nmol/10 aliquots of aliquots 40nmol/40 aliquots 40nmol/40 aliquots 96nmol/96 aliquots 96nmol/96 aliquots Quality control Mass spectrometry MALDI MS and analytical HPLC MALDI MS and analytical HPLC Delivery time§ 2 weeks 3 weeks 3 weeks Shipment Lyophilized at room temp. In solution on wet ice In solution on wet ice † See HeavyPeptide Synthesis Service for all information on HeavyPeptide custom synthesis. § These production times are estimates that vary based on the number of kits ordered.

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 25 targeted quantitation

Measuring expression of selected proteins

Thermo Scientific PEPotec SRM Peptide Libraries Fully synthetic, crude peptides customized for the development of mid- to high-throughput SRM and MRM assays.

The study of proteomes, sub-proteomes and protein pathways often requires quantitative MS analysis that depends on the identification and validation of SRM and MRM assays. PEPotec SRM Peptide Libraries offer great convenience and flexibility in the development of quantitative MS approaches while accelerating biological assays by reducing the setup time of MS experiments. These peptide libraries were developed as a result of our involvement in the SRMAtlas project, which seeks to map the entire human proteome. The standard service supplies a suspension of at least 0.1mg of each crude peptide housed in individual tubes in a 96-well plate format with either arginine (R) or lysine (K) as the C-terminal amino acid. Three quality control grades are available, and optional services and peptide modifications are offered to give you Applications: the peptide libraries that fit your experimental needs. • Mid- to high-throughput development of SRM and MRM assays • MS workflows with relative and absolute quantitation strategies Highlights: • Convenient – peptides are provided in individual 2-D barcoded tubes Includes: in 96-tube plates • Fully synthetic crude (as synthesized) peptides • Application-specific – grades and optional services help you get the right • Multiple grades of QC analysis and optional services and modifications peptides for your assay • Provided in individual Thermo Scientific™ Matrix™ 96-Tube plates • Easy to use – standard libraries are delivered suspended in 0.1% trifluoroacetic acid (TFA) in 50% (v/v) acetonitrile/water • Flexible – extensive list of available modifications

Table 1. Thermo Scientific PEPotec SRM Peptide Libraries – Three grades to fit your experimental needs.

Grade 1 Grade 2 Grade 3 Fast and Easy Greater Analysis Maximum Assurance Quantity >0.1mg Length1 6 to 25 amino acids. Up to 35 amino acids are available for an additional fee Purity Crude (as synthesized) Formulation1 Suspended in 0.1% TFA in 50% (v/v) acetonitrile/water Delivery format Matrix 96-Tube plates (Product # 3712MTX) C-terminal residue1 R or K Counter ion TFA Quality control (QC) MS check of 5% of peptides MS check of 100% of peptides MS analysis of 100% of peptides Peptide resynthesis2 Not provided Not provided One resynthesis provided Failed synthesis policy You pay for entire set of peptides ordered You only pay for peptides synthesized You only pay for peptides synthesized Included documentation Peptide amount Peptide amount Peptide amount and MS spectra Minimum order3 24 peptides 4 peptides 4 peptides 1 Changes to the standard length restrictions, formulation and C-terminal residues are available as optional services. 2 Peptides not detected during MS analysis will be resynthesized (depending on the grade selected). 3 Orders for fewer than 48 peptides incur a plate fee.

26 For more information, or to download product instructions, visit thermoscientific.com/pierce Table 2. Thermo Scientific PEPotec SRM Peptide Library optional services.

QC: Analytical HPLC & MALDI-MS of 100% of samples

QC: LC -MS of 100% of samples

Lyophilized

Individually labeled tubes

Peptides that are 3-5 or 26-35 amino acids in length

Table 3. Thermo Scientific PEPotec SRM Peptide optional modifications – Available with all grades on a per-peptide basis.

C-terminal heavy labeling at R or K Acetylation at side chain of Lysine [Lys(Ac)]

Internal heavy labeling at A,R,I,L,K,F,P or V Methylation at side chain of Lysine [Lys(Me)] and Arginine [Arg(Me)]

Alternative heavy amino acid at C-term Dimethylation at side chain of Lysine [Lys(Me)2]

Alternative light amino acid at C-term 3-Chloro-tyrosine

Phosphorylation at 1–3 site Hydroxyprolinew (Hyp)

All cysteines protected by carbamidomethylation (CAM) Isoaspartic acid

Diglycine ubiquitination motif on Lysine [Lys(GG)] Formylation at C-term

Methionine sulfoxide [Met(O)]

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 27 targeted quantitation

Measuring expression of selected proteins

Thermo Scientific Pierce Peptide Retention Time Calibration Mixture Easy prediction of peptide retention time.

The prediction of peptide retention time is a tool to assess chromatographic performance and to assist in the development of multiplexed, high-throughput mass spectrometric assays. The Thermo Scientific™ Pierce™ Peptide Retention Time Calibration Mixture and Pinpoint Software can be used to predict peptide retention time from sequence alone or to streamline the transition from qualitative protein discovery results to the development of targeted MS assays on Thermo Scientific Triple Quadrupole, Orbitrap, Exactive and Ion Trap Mass Spectrometers. The Pierce Peptide Retention Time Calibration Mixture can be used for optimization and regular assessment of chromatographic performance and for rapid development of multiplexed, scheduled targeted MS assays for the quantification of dozens to hundreds of peptide targets per run.

The Pierce Peptide Retention Time Calibration Mixture contains 15 synthetic Highlights: heavy peptides mixed at an equimolar ratio that elute across the chromatographic • Assess chromatography and MS instrument performance gradient. The peptide sequences and chromatographic results are used to assess • Predict peptide retention across multiple instrument platforms LC performance. In addition, the observed retention times and hydrophobicity • Predict peptide retention time from sequence using calculated factors (HF) for these calibrants are fit to a linear equation to determine the slope hydrophobicity factor of the retention time/HF relationship. This equation and the HF of uncharacterized • Optimize scheduled MS acquisition windows for improved quantification peptides are then used to predict retention time. and increased multiplexing • Normalize results for variation in retention times and peak intensities between runs • Normalize collision energies between instruments and platforms

A.

B. Figure 1. Chromatographic analysis of the Thermo Scientific Pierce Peptide Retention Time Calibration Mixture. A. The Pierce Peptide Retention Time Calibration Mixture (250fmol) was analyzed in duplicate on an LTQ XL Orbitrap Mass Spectrometer using a self-packed column (75µm x 20cm) containing Magic™ C18 (Michrom Bioresources) using a 0.25% per minute gradient of Buffer A (0.1% formic acid) and Buffer B (0.1% formic acid/99.9 % acetonitrile) at 300nL per minute. B. The Pierce Peptide Retention Time Calibration Mixture was also analyzed on a Thermo Scientific™ TSQ Vantage Mass Spectrometer using a Thermo Scientific™ Hypersil™ GOLD C18 column (1.0 x 150mm, Product # 25005-150165) with a 1.0% per minute gradient at 120µL per minute. Numbered peaks correspond to the calibrant peptides described above.

28 For more information, or to download product instructions, visit thermoscientific.com/pierce Table 1. Thermo Scientific Pierce Peptide Retention Time Calibration 105.8 Mixture components and properties. 95.35

The peptide sequences, peptide masses and chromatographic behavior of 84.91 each component of the Pierce Peptide Retention Time Calibration Mixture are given below. The position and identity of the heavy isotope-labeled amino 74.46 acid in each sequence is indicated in bold. 64.02

Peptide Sequence Mass Hydrophobicity 53.57 Factor (HF) 43.13 1 SSAAPPPPPR 985.5220 7.56 32.69

2 GISNEGQNASIK 1224.6189 15.50 (Minutes) Retention Time 3 HVLTSIGEK 990.5589 15.52 22.24 4 DIPVPKPK 900.5524 17.65 11.8 5 IGDYAGIK 843.4582 19.15 1.35 0 20 40 60 80 100 120 6 TASEFDSAIAQDK 1389.6503 25.88 Library Retention Time (Minutes) 7 SAAGAFGPELSR 1171.5861 25.24 Figure 3. Prediction of relative peptide retention times between LC-MS systems. The Pierce 8 ELGQSGVDTYLQTK 1545.7766 28.37 Peptide Retention Time Calibration Mixture was analyzed using LTQ Orbitrap XL and TSQ Vantage 9 GLILVGGYGTR 1114.6374 32.18 Mass Spectrometers. Enzymatic digests from lysed 293T cells treated with 5μg/mL insulin for 10 GILFVGSGVSGGEEGAR 1600.8084 34.50 30 minutes were analyzed on an LTQ Orbitrap XL Mass Spectrometer. Search results from Proteome Discoverer 1.1 Software and the calibrant results from both platforms were imported 11 SFANQPLEVVYSK 1488.7704 34.96 into Pinpoint 1.1 Software to develop scheduled selective reaction monitoring assays for 478 12 LTILEELR 995.5890 37.30 peptides in 35 proteins. Retention times observed on the TSQ Vantage Instrument were plotted 13 NGFILDGFPR 1144.5905 40.42 against the retention times observed on the Orbitrap XL Instrument. 14 ELASGLSFPVGFK 1358.7326 41.18 15 LSSEAPALFQFDLK 1572.8279 46.66

17 Ordering Information 15 Product # Description Pkg. Size

13 88320 Pierce Peptide Retention Time Calibration 50µL Mixture, 0.5pmol/µL

y = 1.019x - 0.196 88321 Pierce Peptide Retention Time Calibration 200µL 11 Mixture, 5pmol/µL R2 = 0.89411 Predicted Time 9

7

5 5.0 7.0 9.0 11.0 13.0 15.0 17.0 Observed (Retention) Elution Time

Figure 2. Retention time prediction with hydrophobicity. The Pierce Peptide Retention Time Calibration Mixture sequences and chromatographic data from Figure 1B were used to calculate the retention time-to-hydrophobicity relationship in Pinpoint 1.1 Software. The observed retention times of 29 tryptic peptides from bovine serum albumin on a TSQ Vantage Mass Spectrometer were plotted against the predicted retention times.

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 29 targeted quantitation

Measuring expression of selected proteins

Thermo Scientific 1-Step Heavy Protein In Vitro Protein Expression Kits Rapid, cell-free expression of recombinant proteins containing stable isotope-labeled (i.e., heavy) amino acids.

The 1-Step Heavy Protein IVT Kit uses a unique HeLa cell lysate supplemented with heavy amino acids for in vitro translation (IVT) of proteins with 90 to 95% isotope incorporation efficiency in less than 8 hours. Heavy proteins expressed using this system can be used as mass spectrometry controls for sample preparation loss, digestion efficiency determination or as quantification standards. Highlights: • Efficient – express heavy proteins with 90 to 95% stable isotope incorporation • Functional – uses the human translational machinery to express more biologically active proteins than other IVT systems The 1-Step Heavy Protein IVT Kit contains all of the necessary components to 13 15 • Flexible – express light proteins or heavy proteins containing C6 N2 L-lysine express a heavy protein including HeLa cell lysate, proprietary accessory proteins, 13 15 and/or C6 N4 L-arginine reaction mix, heavy amino acids, positive-control GFP DNA and the pT7CFE1- • Convenient – perform transcription and translation in a single step CGST-HA-His cloning vector. The benefits of in vitro expression of heavy proteins • High performance – greater yields compared to rabbit reticulocyte in vitro over traditional in vivo systems include expression of toxic or insoluble proteins, translation a more rapid protein synthesis, and a more economical use of heavy amino acids compared to stable-isotope labeled cell lines. This small-scale expression Applications: method makes it easy to express numerous heavy proteins simultaneously or to • Identify ideal peptides for targeted quantitation express large quantities of a single heavy protein up to 100µg/mL. Tandem affinity • Verify protein digestion efficiency purification of heavy proteins is aided using an expression vector containing multiple • Control for protein sample preparation variability and affinity enrichment loss affinity tags including GST, HA and 6xHis.

Cell Lysate Protein Purification and Digestion

Reaction Mixture: • labeled amino acids • accessory proteins • energy mix, salts LC-MS • nucleotides

Vector IVT DNA

Expressed Light Heavy Isotope Protein Incorporation Figure 1. Workflow for heavy recombinant protein expression, purification and mass spectrometry analysis. Cell lysates are Relative Determination Intensity combined with the reaction mixture, vector DNA and stable isotope- labeled amino acids to express recombinant proteins. Expressed proteins are then purified and digested into peptides for LC-MS analysis. m/z

30 For more information, or to download product instructions, visit thermoscientific.com/pierce 100 100 90 90 80 80 ) 70 70 /mL g

(µ 60 60 on

ti 50 50 ra 40 40 ent

nc 30 30 Co Isotope Incorporation (%) 20 20 10 10 Figure 2. Heavy green-fluorescent protein (GFP) expression. 0 0 GFP expression with time, showing corresponding heavy-isotope incorporation. 0 1 2 3 4 5 6 Incubation Time (hours)

A. B. Figure 3. Expression of heavy mammalian proteins. Panel A: Protein Mass Isotope Western blot of GST-fusion proteins GFP, BAD, cyclinD1, retinoblastoma Sample (kDa) Incorp. (RB), p53 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) GFP 27 95% expressed using human IVT extract (the arrow indicates an anti-GST cross-reacting band in the lysate). Panel B: Isotope incorporation from BAD 19 92% peptides derived from the proteins in Panel A. Cyclin D1 36 97% Rb 110 96% p53 53 91% GAPDH 37 94%

GFP BAD Cyclin Rb p53 GAPDH D1

Ordering Information

Product # Description Pkg. Size 88330 1-Step Heavy Protein IVT Kit 8-rxn set Sufficient for eight reactions of 25µL each Kit Contents: HeLa Lysate 110µL Accessory Proteins 25µL Reaction Mix 40µL 13 15 C6 N4 L-Arginine 25µL 13 15 C6 N2 L-Lysine 25µL Positive Control DNA: pCFE-GFP 10µg pT7CFE1-CGST-HA-His 10µg Nuclease-Free Water 1.5mL 88331 1-Step Heavy Protein IVT Kit 40-rxn set Sufficient for 40 reactions of 25µL each Kit Contents: HeLa Lysate 500µL Accessory Proteins 100µL Reaction Mix 200µL 13 15 C6 N4 L-Arginine 25µL 13 15 C6 N2 L-Lysine 25µL Positive Control DNA: pCFE-GFP 10µg pT7CFE1-CGST-HA-His 10µg Nuclease-Free Water 1.5mL

For a list of references using the 1-Step Heavy Protein IVT Kit, please see the back cover.

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 31 targeted quantitation

Measuring expression of selected proteins

Table 1. Thermo Scientific Mass Spectrometry systems and capabilities. Discovery Proteomics Isobaric Mass Tags (Data Dependent Mass (iTRAQ 4™, TMT6plex, Isobaric Mass Tags Targeted Spectrometry - Protein Instrument SILAC/iCAT™/Mass Tags iTRAQ8™) (TMT10plex) Quantitation Indentification) LTQ Velos No Yes (PQD) No Yes (tSIM, SRM) Good LTQ Velos Pro No Yes (PQD No Yes (tSIM, SRM) Good LTQ XL ETD No Yes (PQD) No Yes (tSIM, SRM) Good MALDI LTQ XL No Yes (PQD) No Yes (tSIM, SRM) Good

LTQ Orbitrap Velos Yes Yes (PQD and HCD) Yes† Yes (HR/AM MS1 & MSn) Excellent LTQ Orbitrap XL ETD Yes Yes (PQD and HCD) Yes† Yes (HR/AM MS1 & MSn) Excellent LTQ Orbitrap XL Yes Yes (PQD and HCD) Yes† Yes (HR/AM MS1 & MSn) Excellent MALDI LTQ Orbitrap XL Yes Yes (PQD and HCD) Yes† Yes (HR/AM MS1 & MSn) Good LTQ Orbitrap Discovery Yes Yes (PQD only) No Yes (HR/AM MS1 & MSn) Good LTQ Orbitrap Velos Pro Yes Yes (PQD and HCD) Yes Yes (HR/AM MS1 & MSn) Excellent Orbitrap ELITE Yes Yes (PQD and HCD) Yes Yes (HR/AM MS1 & MSn) Excellent Orbitrap Fusion Tribrid Yes Yes (HCD) Yes Yes (HR/AM MS1 & MSn) Excellent

Exactive Yes No No Yes (HR/AM MS1) N/A Exactive plus Yes No No Yes (HR/AM MS1) N/A Q Exactive Yes Yes (HCD only) Yes Yes (HR/AM MS1 & MSn) Excellent Q Exactive plus Yes Yes (HCD only) Yes Yes (HR/AM MS1 & MSn) Excellent

LTQ FT Ultra Yes No No Yes (HR/AM MS1 & MSn) Good

TSQ Access Max No No No Yes (SRM) Not recommended TSQ Quantam Ultra No No No Yes (SRM) Not recommended TSQ Vantage No No No Yes (SRM) Not recommended TSQ Endura No No No Yes (SRM) Not recommended TSQ Quantiva No No No Yes (SRM) Not recommended Please note: † Can be done but with very slow duty cycle. PQD option included in all instruments via the Xcalibur software tSIM = Targeted Selective Reaction Monitoring HR/AM = High Resolution/Accurate Mass SRM = Selective Reaction Monitoring

32 For more information, or to download product instructions, visit thermoscientific.com/pierce related products

Ordering Information

Abundant Protein Depletion Protein Digestion – Mass Spec Grade Proteases, Reagents and Kits

Product # Description Pkg. Size Product # Description Pkg. Size 85160 Albumin Depletion Kit 24-rxn kit 90057 Trypsin Protease, MS Grade†† 5 x 20μg 85161 Top 2 Abundant Protein Depletion 6 columns 90058 Trypsin Protease, MS Grade†† 5 x 100μg Spin Columns† †† 85162 Top 2 Abundant Protein Depletion 24 columns 90059 Trypsin Protease, MS Grade 1mg † Spin Columns 90305 Trypsin Protease, MS Grade, Frozen Liquid 100μg 85164 Top 12 Abundant Protein Depletion 6 columns 90300 LysN Protease, MS Grade 20μg Spin Columns† 85165 Top 12 Abundant Protein Depletion 24 columns 90301 LysN Protease, MS Grade 5 x 20μg † Spin Columns 90051 LysC Protease, MS Grade 20μg 90053 AspN Protease, MS Grade 2μg Protein Desalting 90054 GluC Protease, MS Grade 5 x 10μg 89877 Zeba™ Micro Spin Desalting Columns, 25 columns 7K MWCO, 75μL 90056 Chymotrypsin (TLCK treated), MS Grade 4 x 25μg 89882 Zeba Spin Desalting Columns, 25 columns 89871 In-Gel Tryptic Digestion Kit Kit 7K MWCO, 0.5mL 89895 In-Solution Tryptic Digestion Kit 89890 Zeba Spin Desalting Columns, 25 columns and Guanidination Kit 7K MWCO, 2mL 89892 Zeba Spin Desalting Columns, 25 columns 84840 Mass Spec Sample Prep Kit for Cultured Cells 20-rxn kit 7K MWCO, 5mL 84841 Digestion Indicator for Mass Spectrometry 10μg 89894 Zeba Spin Desalting Columns, 25 columns 7K MWCO, 10mL 89807 Zeba 96-well Spin Desalting Plates, 2 plates Phosphopeptide Enrichment and Clean-Up 7K MWCO 88300 Pierce™ Fe-NTA Phosphopeptide 30-rxn kit Enrichment Kit 87764 Zeba Micro Spin Desalting Columns, 25 columns ™ 40K MWCO, 75μL 88301 Pierce TiO2 Phosphopeptide Enrichment 24-rxn kit and Clean-Up Kit 87766 Zeba Spin Desalting Columns, 25 columns ™ 40K MWCO, 0.5mL 88811 Pierce Magnetic TiO2 Phosphopeptide 96-rxn kit Enrichment Kit 87769 Zeba Spin Desalting Columns, 25 columns 40K MWCO, 2mL 88812 Pierce Magnetic TiO2 24-rxn kit Phosphopeptide Enrichment Kit 87771 Zeba Spin Desalting Columns, 25 columns 40K MWCO, 5mL 88302 Pierce™ Graphite Spin Columns, 0.5mL 30 columns 87773 Zeba Spin Desalting Columns, 25 columns 40K MWCO, 10mL Peptide Clean-Up 87774 Zeba 96-well Spin Desalting Plates, 2 plates 87776 Pierce™ Detergent Removal Spin Column, 25 columns 40K MWCO 125μL 87777 Pierce Detergent Removal Spin Column, 25 columns Active Site Protein Labeling and Enrichment 0.5mL 88310 Pierce™ Kinase Enrichment Kit with 16-rxn kit 87778 Pierce Detergent Removal Spin Column, 2mL 5 columns ATP Probe 87779 Pierce Detergent Removal Spin Column, 4mL 5 columns ™ 88311 ActivX Desthiobiotin-ATP Probe 16 x 12.6μg ™ 87780 Pierce Detergent Removal Resin 10mL ™ 88312 Pierce Kinase Enrichment Kit with 16-rxn kit ™ ADP Probe 88304 Pierce Detergent Removal Spin Plates 2 plates 88313 ActivX™ Desthiobiotin-ADP Probe 16 x 9.9μg 88305 HiPPR Detergent Removal Spin Column Kit 5mL kit (Resin + Columns) 88314 Pierce™ GTPase Enrichment Kit with 16-rxn kit GTP Probe 88306 HiPPR Detergent Removal Spin Columns, 0.1mL 24 columns 88315 ActivX Desthiobiotin-GTP Probe 16 x 12.9μg 88307 HiPPR Detergent Removal 96-well Spin Plates 2 plates ™ 88316 ActivX Azido-FP Serine Hydrolase 3.5μg 89870 Pierce C18 Spin Columns 25 columns Probe 87782 Pierce C18 Tips, 10μL bed 96 tips 88317 ActivX Desthiobiotin-FP Serine 4.6μg 87784 Pierce C18 Tips, 100μL bed 96 tips Hydrolase Probe 84850 Pierce C18 Spin Tips 96 tips

88318 ActivX TAMRA-FP Serine Hydrolase 6.8μg † † † Probe Limit one pack per order. Limit five packs per order. † Limit one pack per order † † Limit five packs per order

To order, call 800.874.3723 or 815.968.0747. Outside the U.S., contact your local branch office or distributor. 33 thermoscientific.com/pierce

SILAC references HeavyPeptide Reagent references 1. Amanchy, R., et al. (2005). Science STKE 267:1-20. 1. Anderson, et al. (2009). Mol. Cell Proteomics 8(5):883-886. 2. Blagoev, B., et al. (2004). Nat. Biotechnol 22(9):1139-1145. 2. Streit, F., et al. (2002). Clin. Chem. 48:955-958. 3. Everley, P., et al. as presented at The American Society for Biocehmistry 3. Anderson, et al. (2002). Mol. Cell. Proteomics 1:845-867. and Molecular Biology. MCP Papers in Press. Published on July 11, 2007 4. Junichi Kamiie, et al. (2008). Pharm. Res. 25(6):1469-1483. as Manuscript M700057-MCP200. 5. Tuthill, C.W., et al. (2000). AAPS PharmSciTech 1:E11. 4. Kratchmarova, I., et al. (2005). Science 308(5727):1472-1477. 6. Desiderio, D.M., et al. (1981). J. Chromatogr. 217:437-452. 5. Mann, M. (2006). Nat. Rev. Mol. Cell Biol.7(12):952-958. 7. Arsene, C.G., et al. (2008). Anal. Chem. 80(11):4154-4160. 6. Ong, S. E., et al. (2002). Mol. Cell Proteomics 1(5):376-386. 8. Barr, J.R. and Maggio, V.L. (1996). Clin. Chem. 42:1676-1682. 7. Selbach, M. and Mann, M. (2006). Nat. Methods 3(12):981-3. 9. Gerber, S.A., et al. (2003). PNAS USA 100(12):6940-6945. 8. Everly, P.A., et al. (2004). Mol. & Cell Proteomics. 3.7:729-735. 10. Kuster, B., et al. (2005). Nat. Rev. Mol. Cell Biol 6:577-583. 9. Levine, A.J. (1997). Cell 88:323-331. 11. Prakash, A., et al. (2009). J. Proteome Res. 6:2733-2739. 12. Yu, Y., et al. (2009). PNAS USA 106:11606-11610. TMT references 13. Malmstrom J., et al. (2009). Nature 460:762-765. 1. Dephoure, N., et al. (2013). Mol. Biol. Cell 24:535-542. 14. Lange, V., et al. (2008). Molecular Systems Biology 4:222. 2. Matsui, K., et al. (2013). Appl. Envir. Microbiol. 79:6576-6584. 15. Arsene, C.G., et al. (2008). Anal. Chem. 80:4154-4160. 3. Xiao, D., et al. (2013). Cardiovasc. Res. 10:1093/cvr/cvt264. 16. Kirsch, S., et al. (2007). J. Chromatography 1153:300-306. 4. Li, N., et al. (2013). Nucleic Acids Res. 41:7073-7083. 17. Guan, F., et al. (2007). Anal. Chem. 79:4627-4635. 5. Islam, K., et al. (2013). PNAS 110:16778-16783. 18. Desiderio, D.M. (1999). J. Chromatography B 731:3-22. 6. Zhuang, G., et al. (2013). Sci. Signal. 6:ra25. 19. Burkit, W.I., et al. (2008). Anal. Chem. 376:242-251. 7. Nawrocki, S., et al. (2013). Clin. Cancer Res. 19:3577-3590. 20. Holzmann, J., et al. (2009). Anal. Chem. 81:10254-10261. 8. Yang, F., et al. (2013). Mol. Cell. Proteomics 12:2497-2508. 21. Kubota K., et al. (2009). Nat. Biotechnol. 10:933-940. 9. León, I., et al. (2013). Mol. Cell. Proteomics 12:2992-3005. 22. Wepf, A., et al. (2009). Nat. Methods 6:203-205. 10. Tang, X., et al. (2013). Occup. Environ. Med. 70:561-567. 11. King, J., et al. (2013). Mol. Biol. Cell 24:2714-2726. 1-Step Heavy Protein In Vitro Protein Expression Kit references 12. Tilghman, S., et al. (2013). Mol. Cell. Proteomics 12:2440-2455. 1. Mikami, S., et al. (2006). Protein Expr. Purif. 46(2):348-57. 13. Sherwood, V., et al. (2013). Carcinogenesis 10.1093/carcin/bgt390. 2. Mikami, S., et al. (2008). Protein Expr. Purif. 62(2):190-8. 14. Bloem, E., et al. (2013). J. Biol. Chem. 288:29670-29679. 3. Hanke S., et al. (2008). J. Proteome Res. 7(3):1118-30. 15. Guo, Q., et al. (2013). Nucleic Acids Res. 41:2609-2620. 4. Ciccimaro E.., et al. (2009). Anal. Chem. 81(9):3304-13. 16. Zhang, X., et al. (2013). Mol. Cell. Biol. 33:1657-1670. 5. Stergachis, A., et al. (2011). Nat. Meth. 8(12):1041-1043. 17. Castro-Núñez, L., et al. (2013). J. Biol. Chem. 288:393-400. 18. Fratini, F., et al. (2012). Neurology 79:1012-1018. 19. Bloem, E., et al. (2012). J. Biol. Chem. 287:5775-5783. 20. Mirza, S. (2012). Circ. Cardiovasc. Genet. 5:477. 21. Weston, A., et al. (2012). Mol. Cell. Proteomics 11:M111.015487. 22. Bennett, E., et al. (2012). Mol. Cell. Proteomics 11:1523-1528. 23. Bailey, D., et al. (2012). PNAS 109:8411-8416. 24. Cargnello, M., et al. (2012). Mol. Cell. Biol. 32:4572-4584. 25. Sánchez-Quiles, V., et al. (2011). Mol. Cell. Proteomics 10: M111.009126. 26. Van Eyk, J. (2011). Circ. Res. 108:490-498. 27. McAlister, G., et al. (2011). Mol. Cell. Proteomics 10:O111.009456. 28. Jedrychowski, M., et al. (2011). Mol. Cell. Proteomics 10:M111.009910. 29. Diaz Vivancos, P., et al. (2011). Plant Physiology 157:256-268.

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