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, and/or HCD fragmentation.
.jpg)
Literature Highlights
Quantitative mass spectrometry in proteomics: a critical review
Bantscheff M, Schirle M, et al.
Anal Bioanal Chem. 2007 Oct;389(4):1017-31.
Download the brochure on Thermo Scientific Solutions for Quantitative Proteomics
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
Quantitative mass spectrometry in proteomics: a critical review
Bantscheff M, Schirle M, et al.
Anal Bioanal Chem. 2007 Oct;389(4):1017-31.
Related Products
Download a comprehensive
Mass Spectrometry Sample Preparation Handbook.
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, 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.
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 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
1. Thermo Scientific SIEVE Software for Differential Expression Analysis: Automated, label-free, semi-quantitative analysis of proteins, peptides, and metabolites based on comparisons of LC/MS and GC/MS data
2. Thermo Scientific Proteome Discoverer: Mass Informatics Platform for Protein Scientists
Orbitrap of Choice for Label-Free Relative Quantitation
The simultaneous high resolution (>100,000) and spectral dynamic range afforded by Thermo Scientific Orbitrap technology enable label-free quantitation by providing essential discrimination between isobaric co-eluting ions and separating analyte signal from noise. High mass accuracy, fast data acquisition, and MS/MS capabilities are also essential to successful label-free quantitation. The Thermo Scientific Orbitrap Elite, Orbitrap Velos Pro, LTQ Orbitrap XL and Q Exactive instruments all have the capabilities necessary for label-free relative quantitation of proteins.
The Orbitrap Elite
- The Orbitrap Elite, a hybrid ion trap-Orbitrap instrument provides the high resolving power (up to 240,000 at m/z 400) required for accurate label-free precursor-based quantitation analyses without compromising fast data acquisition (1).
- The unmatched resolution and mass accuracy of the Orbitrap Elite MS enables sensitive precursor peak detection and provides essential discrimination between co-eluting isobaric ions. It also separates analyte signal from noise, leading to the most accurate and precise quantitation.
- Identification of the peptides in label-free quantitation experiments is not restricted by fragmentation type. CID, ETD, and/or HCD fragmentation can be applied separately or in combination which, because the fragmentation techniques are complementary, can lead to the identification of more peptides.
References
1. Ultra high resolution linear ion trap Orbitrap mass spectrometer (Orbitrap Elite) facilitates top down LC MS/MS and versatile peptide fragmentation modes
Michalski A, Damoc E, et al.
Mol Cell Proteomics. 2012 Mar;11(3):O111.013698.
