mass spectrometry Thermo Scientific Solutions for Quantitative Proteomics A leader in quantitative proteomics, we supply complete solutions that redefine the limits of quantitative proteomics and help you exceed your research goals. Part of Thermo Fisher Scientific Quantitative Proteomics Redefining Quantitative Proteomics TO UNDERSTAND the functions of individual proteins Discovery-based analyses identify and their place in complex biological systems, it is often and quantify necessary to measure changes in protein abundance With discovery-based quantitative analyses, the goal is relative to changes in the state of the system. As such, to both identify proteins and measure their abundance modern proteomics has evolved from an almost changes across multiple sample sets. Several discovery- exclusively qualitative technique into one that spans a based techniques have been developed, including continuum of qualitative and quantitative approaches. stable isotope labeling by amino acids in cell culture Traditionally, quantitative measurements of protein (SILAC), chemical labeling with isobaric mass tags, abundance were done by Western blotting. However, and label-free.1, 2, 3 Western blotting almost always requires a priori knowledge of the system and the expected changes in Targeted analyses are fast and sensitive order to obtain an appropriate target antibody. Antibodies Targeted analyses can be performed once candidate are often not available, not specific, or very expensive. proteins are identified by either discovery-based MS Antibodies for site-specific modifications can be even experiments or alternate, a priori information. Target ed more difficult to obtain. In addition, Western blotting is analyses provide improved quantitation sensitivity (LOQ) sample intensive, has limited linear dynamic range, and, combined with increased speed of analyses, enable and typically only a single target is quantified in each analysis of expanded sample sets to assess the validity Western blot. of the candidate proteins. Spiking biological samples Alternatively, liquid chromatography coupled to with proteotypic, isotopically labeled peptide standards mass spectrometry (LC-MS) has emerged as a powerful makes possible the absolute quantitation of each technology for system-wide identification and quantitation protein or post-translational modification of interest. of proteins. It can be used for both discovery-based (untargeted) and targeted determination of changes Choosing the most appropriate quantitative proteomics in protein abundance. It can also be used to measure technique depends on experimental demands and changes in the abundance of protein-specific post- instrumental capabilities. This brochure is intended to translational modifications (PTMs), facilitating location assist the decision-making process. of the modified residue. Quantitative MS analyses require less sample than Western blotting and can detect and quantify multiple analytes in a single experiment. Number Samples Precision Technique Type (per LC-MS run) (% CV) Benefits Drawbacks Instruments Discovery-based (untargeted quantitative analysis coupling protein identification with quantitation) Label- Relative 1 < 30 • Applicable to any sample type • Each sample runs individually (low throughput) Orbitrap Free • Cost-efficient sample preparation • Requires extremely reproducible LC separations • Minimal sample handling • Requires multiple technical replicates TMT Relative 2 to 6 < 20 • Applicable to any sample type • Requires extensive fractionation or long Orbitrap • Multiplexing increases MS throughput chromatographic gradients with HCD Ion Trap with PQD or Trap-HCD SILAC Relative 2 or 3 < 20 • Least susceptible to inter-sample variations in • Only readily applicable to cell cultures Orbitrap sample handling and preparation • Increases MS spectral complexity • Multiplexing increases MS throughput Targeted (analysis of predetermined peptides from discovery-based experiments or literature) HR/AM- Relative 1 < 20 • Uses the same MS system as discovery quantitation • Requires reproducible LC separations Orbitrap SIM or • Easy method development using Pinpoint software Absolute iSRM Relative 1 < 10 • Up to 15,000 SRM transitions per run • Requires reproducible LC separations TSQ triple or • Simultaneous protein quantitation and confirmation quadrupole Absolute of identity • Suitable for determining the absolute quantity of a protein in a complex biological matrix • Easy method development using Pinpoint software Discovery-based and targeted quantitation techniques all have specific applications and advantages 2 SILAC Improves the Throughput of Discovery-Based Quantitative MS Analyses and Increases Accuracy of Results STABLE-ISOTOPE LABELING BY AMINO ACIDS form. Because labeled and unlabeled samples are KEY POINTS IN CELL CULTURE (SILAC) is a powerful and widely combined during the initial steps of sample preparation, used method of identifying and quantifying relative SILAC minimizes the quantitative error inherent in Advantages: changes in complex protein samples. It can be applied to handling separate samples in parallel. In addition, the • 2-3 samples complex biomarker discovery and systems biology studies mixing of samples permits a variety of enrichment • Reduced sample as well as to isolated proteins and protein complexes. techniques including immunoprecipitation. These can handling decreases As its name implies, SILAC involves labeling protein improve the detection of abundance changes for both variability samples in vivo by substituting an isotopically heavy low-abundance proteins and post-translational • Data analysis using form of an amino acid for the naturally occurring light modifications such as phosphorylation or glycosylation. Proteome Discoverer software Disadvantages: SILAC WORKFLOW • Cell cultures only Cell Cultures • High-resolution MS 2-3 samples grown in light- and heavy-labeled amino acid media (FT or Orbitrap technology) required Thermo Scientific Pierce SILAC protein quantitation kits contain all of the reagents needed for comparing two or three sample types in a wide variety of mammalian cell lines. They are compatible with many Thermo Scientific protein/peptide enrichment technologies. 3 Quantitative Proteomics TMT Labeling Increases the Number of Samples Analyzed, and Peptides Identified and Quantified, in a Single Analysis ISOBARIC CHEMICAL TAGS are a more universal TMT WORKFLOW alternative to SILAC for simultaneous identification and Cell Culture or Tissue quantitation of proteins in multi-sample sets. They 2-6 samples can facilitate relative quantitation of a wide variety of samples including cells, tissues, and biological fluids. Thermo Scientific Tandem Mass Tag (TMT) reagents are isobaric mass tags consisting of an MS/MS reporter group, a spacer arm, and an amine-reactive KEY POINTS group. Amine-reactive groups covalently bind to peptide Advantages: N-termini or to lysine residues. Each tag fragments • 2-6 samples during MS/MS, producing unique reporter ions. • Any sample type Protein quantitation is accomplished by comparing the intensities of the reporter ions. • Ion trap- or Orbitrap- based instruments • Data analysis using Proteome Discoverer software High-resolution HCD-MS/MS improves quantitative precision in TMT® analyses The ability to generate low-m/z reporter ions and to distinguish them from isobaric interferences is essential for consistent, precise TMT 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. TMT kits are available in 2-plex for comparing two samples in small profiling studies. They are also available in 6-plex for comparing up to six samples in complex analyses with multiple conditions (e.g. time courses, dose responses, replicates, and multiple-sample comparisons). TMT kits can be combined with many Thermo Scientific peptide enrichment technologies. 4 Label-Free Quantitation Enables Exploration of the Proteome in Greater Depth LABEL-FREE DIFFERENTIAL ANALYSIS has gained Each of the highly complex samples found in a typical KEY POINTS popularity for discovery-based quantitative proteomics. label-free differential analysis study is run individually. It requires no specific sample preparation and accom- Therefore, meticulous sample handling, sample prepa- Advantages: modates large numbers of diverse samples. Label-free ration, reproducible chromatography between technical • Unlimited number quantitation involves extracting peptide chromatograms and biological replicates, and sensitive, high-resolution, of samples from LC-MS/MS runs and integrating peak areas over accurate-mass MS are all essential. Thermo Scientific • Any sample type the chromatographic time scale. Typically favored for solutions for label-free differential analysis are complete • Data analysis using shotgun proteomics, the label-free approach has been and integrated. Thermo Scientific EASY-nLC systems SIEVE software incorporated into large-scale biomarker discovery studies use a split-free design to achieve exceptional stability Disadvantages: measuring disease-related changes. It has demonstrated and reproducibility. They easily couple to all Thermo • Multiple technical high reproducibility and linearity at both peptide and Scientific mass spectrometers, including the Orbitrap- replicates protein levels.3 based systems that have the high resolution necessary • Reproducible for successful label-free quantitation. chromatography Thermo Scientific SIEVE software enables