Discovery-based relative quantification is an analytical approach that allows the scientist to determine relative protein abundance changes across a set of samples simultaneously and without the requirement for prior knowledge of the proteins involved.
To understand the functions of individual proteins and their place in complex biological systems, it is often necessary to measure changes in protein abundance relative to changes in the state of the system. These measurements have traditionally been performed using Western blot analyses. More recently, modern proteomics has evolved to include a variety of technologies for the routine quantitative analyses of both known and unknown targets. Discovery-based relative quantification is an analytical approach that allows the scientist to determine relative protein abundance changes across a set of samples simultaneously and without the requirement for prior knowledge of the proteins involved. Here we describe three commonly used techniques for relative quantitation of unknown protein/peptide targets using mass spectrometry:
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Overview
Workflow Overview for Label-Free Relative Quantitation
Label-free relative quantitation involves comparing the abundances of proteins in multiple samples without the use of isotopic labels. Samples are run individually, then common chromatographic features are used to align the various runs with software. Signals corresponding to individual peptide ions are integrated over the LC time scale, and compared between runs. Label-free analysis is a powerful and widely used technique for identifying and quantifying relative changes in complex protein samples. It can be applied to complex biomarker discovery and systems biology studies as well as to isolated proteins and protein complexes. Key benefits of label-free precursor-based quantitation include the fact that unlimited numbers of samples can be compared, samples can be of any origin, and identification of the peptides is not restricted by fragmentation technique, allowing use of CID, ETD, EThcD and/or HCD fragmentation.
Sample Preparation
Sample Prep Workflow for Label-Free Relative Quantitation
Label-free quantitation requires no specific sample preparation (ie. labeling) and accommodates large numbers of diverse samples. Because of this, label-free quantitation is typically favored for bottom-up “shotgun” proteomics and has been incorporated into large-scale biomarker discovery studies measuring disease-related changes. Because each sample is run individually, however, and because samples are typically extremely complex, all conditions up to and including LC analysis must be highly reproducible. Therefore, meticulous sample handling, sample preparation, reproducible chromatography between technical and biological replicates, and sensitive, high-resolution, accurate-mass MS are all essential. Offline fractionation is not recommended due to the negative effect on quantitation accuracy resulting from slight variations in sample handling.
Regardless of the origin of the protein(s) to be analyzed, a key step in preparation of samples for label-free quantitation is the generation of peptides suitable for MS analysis. Peptide preparation involves reduction and alkylation of cysteines, digestion of the sample into peptides, desalting and concentration of the peptides.
Trypsin is by far the most commonly used digestion enzyme. It cuts C-terminally to arginine and lysine residues, when not blocked by an adjacent proline residue. This has the advantage of generating peptides that are of moderate size due to the natural abundance rates of these amino acids and that tend to carry two or three positive charges when ionized by electrospray. Tryptic peptides are generally optimal for MS/MS analysis via collision-induced dissociation as their charge state and length provide ready fragmentation yielding information-rich, but not overly complex spectra. Alternative enzymes, such as GluC, AspN, and LysC, are sometimes used when larger peptides are required or when a region of interest within a given protein will not yield a suitable tryptic peptide.
Resources
Mass Spec Analysis Technical Handbooks
Sample Prep Tools for Mass Spectrometry
Related Products
Protein Digestion for Mass Spectrometry
Mass Spectrometry
Mass Spectrometry Workflow for Label-Free Relative Quantitation
Quantitation results from label-free experiments are based on the relative precursor intensities of each peptide across multiple runs. Because of this, high-resolution LC separation should be used to reduce the number of co-eluting species, and all LC runs must be highly reproducible between technical and biological replicates. Also imperative are high resolution and accurate mass analyses to resolve the co-eluting isobaric species to reduce quantitation interference. This is especially important for samples of high complexity and/or high dynamic range.
For label-free peptide quantitation, columns at least 15 cm in length with LC gradients greater than one hour and optimal sample loads are recommended in order to achieve base-peak separation, reproducibility and minimize overlapping precursor ions.
Thermo Scientific Pierce Peptide Retention Time Calibration (PRTC) mixture helps optimize and assess LC parameters, identify total peptide elution window and optimize MS parameters. Including it in quantitative samples makes it possible to monitor and normalize LC-MS performance between samples and over time.
A key benefit to precursor-based quantitation, such as SILAC and label-free quantitation, is that identification of the peptides is not restricted to a particular fragmentation technique. CID, ETD, EThcD, and HCD fragmentation can be applied separately or in combination which, because the fragmentation techniques are complementary, can lead to the identification of more peptides.
Resources
System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot...
Nagaraj N, Kulak NA, et al.
Mol Cell Proteomics. 2012 Mar;11(3):M111.013722.
Performance of a linear ion trap-Orbitrap hybrid for peptide analysis.
Yates JR, Cociorva D, et al.
Anal Chem. 2006 Jan 15;78(2):493-500.
A Quadrupole-Orbitrap Hybrid Mass Spectrometer Offers Highest Benchtop Performance for In-Depth Anal...
Hao Z, Zhang Y, et al.
Application Note 552
The Q Exactive HF, a Benchtop Mass Spectrometer with a Pre-filter, High-performance Quadrupole and a...
Scheltema RA, Hauschild J, Lange O, Hornburg D, et al.
Mol Cell Proteomics 2014, 13(12), 3698-3708.
Rapid and Deep Proteomes by Faster Sequencing on a Benchtop Quadrupole Ultra-High-Field Orbitrap Mas...
Kelstrup CD, Jersie-Christensen R.R, Batth TS, et al.
J Proteome Res 2014, 13 (12), 6187-6195.
The one hour yeast proteome
Hebert AS, Richards AL, Bailey DJ, Ulbrich A, Coughlin EE, Westphall MS, Coon JJ.
Mol Cell Proteomics 2014 13(1), 339-47.
Pushing the Limits of Bottom-Up Proteomics with State-Of-The-Art Capillary UHPLC and Orbitrap Mass S...
Lopez-Ferrer D, Blank M , Meding, et al.
Application Note 639
Standardized Workflows for Near Complete Proteome Identification
Jiang X, Xu S, Wang Z, et al.
Scientific Poster
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Data Analysis
Label-Free Relative Quantitation Data Processing Workflow
Thermo Scientific SIEVE software is a key facilitator of label-free protein quantitation (1). It enables researchers to analyze label-free data according to common, predefined experimental designs such as two-group randomized controlled studies or single-group longitudinal studies. Trend analysis can be performed to detect changes associated with dosage effects or time-points for the comparison of multiple classes of samples. Protein identification using the accurate mass precursor information and the CID, HCD or ETD fragmentation data is done using Thermo Scientific Proteome Discoverer software (2).
For detailed step-by-step information about label-free quantitative analysis, using SIEVE™ and Proteome Discoverer™ software, and to download a free 60-day demonstration version of both softwares, visit the Thermo Scientific Proteomics Software Portal.
After preliminary identification and validation of data sets, scientists face the critical, time-consuming task of interpreting their proteomics data — extracting meaningful biological information from multiple, complex data sets. Thermo Scientific ProteinCenter software is a web-based data interpretation tool that enables scientists to compare and interpret data sets in minutes instead of months. Scientists can now:
- Explore the biological context of a single protein
- Explore the biological context of data sets
- Explore biological context of data set comparisons - independent of search engine and database
- Explore biological context in quantitative studies
ProteinCenter™ software enables filtering, clustering and statistical bioinformatics analysis utilizing a regularly updated, consolidated protein-sequence database containing >13 million non-redundant proteins.
For more information on ProteinCenter software, please visit the Thermo Scientific Proteomics Software Portal.
References
Thermo Scientific Proteome Discoverer: Mass Informatics Platform for Protein Scientists
Data Analysis Workflow for Label-Free Relative Quantitation
Thermo Scientific SIEVE software is a key facilitator of label-free protein quantitation (1). It enables researchers to analyze label-free data according to common, predefined experimental designs such as two-group randomized controlled studies or single-group longitudinal studies. Trend analysis can be performed to detect changes associated with dosage effects or time-points for the comparison of multiple classes of samples. Protein identification using the accurate mass precursor information and the CID, HCD, EThcD or ETD fragmentation data is done using Thermo Scientific Proteome Discoverer software (2).
For detailed step-by-step information about label-free quantitative analysis, using SIEVE™ and Proteome Discoverer™ software, and to download a free 60-day demonstration version of both softwares, visit the Thermo Scientific Proteomics Software Portal.
References
2. Thermo Scientific Proteome Discoverer: Mass Informatics Platform for Protein Scientists
Grant Central
Grant Central Resources for Label-Free Relative Quantitation
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Pushing the Limits of Bottom-Up Proteomics with State-Of-The-Art Capillary UHPLC and Orbitrap Mass S...
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The One Hour Yeast Proteome
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