Label Free Quantitation

Principle:

Label-free methods for both relative and absolute quantitation have been developed as a rapid and low-cost alternative to other quantitative proteomic approaches. (Relative quantitation hereafter)

In label free quantitation, protein profiling comparisons are based on the relative intensities of extracted ion chromatograms from enzymatic digests. This approach therefore, does not require any metabolic, chemical, enzymatic labeling or premixing of the samples to be compared multiple datasets, typically run in triplicate, are aligned using mass and LC elution time. (Figure1)

Label free protein expression utilizes a bottom-up proteomic strategy. Bottom-up proteomics differs from top down approaches (i.e. 2D-DIGE) as they analyze a proteome at the peptide level. One advantage of bottom up proteomics is increased overall proteome coverage. Moreover, most bottom up methods simultaneously provide both qualitative and quantitative data in a single run which improves throughput, and quantifying at the peptide level leads directly into a bottom up confirmation/validation analysis thereby avoiding the peptide selection step in this procedure.

This method observes all detectable peptides and, if interrogated by MS/MS, their corresponding fragment ions. This approach quantifies a peptide by its intensity and groups each peptide across individual samples based on its accurate mass and retention time. Once compiled, these intensities associated with accurate mass and retention time are organized into a peptide array which can be subsequently analyzed by statistical techniques that accommodate high dimensional data. As with other bottom up approaches, this method is suited to pre-fractionation strategies but it is not limited in the comparisons it can make as isotope labels are not used. Therefore, this is a very attractive alternative to bottom up label and top down proteomic platforms when analyzing samples from complex experimental designs such as those derived from clinical studies.

Equipment:

The approach requires excellent mass accuracy and reproducible chromatography, thus, the mass spectrometer platforms of choice for this procedure are the LTQ Orbitrap XL and the Bruker Apex Qe 9.4T FT-ICR. Each of these mass spectrometric platforms routinely performs with mass accuracies of better than 3 ppm. The LTQ Orbitrap XL is equipped with a Waters nanoACQUITY which has superb chromatography reproducibility of ±0.2% minute elution time. Data analysis is done later.

Amount:

Recommended sample amount is 5 to 25µg; each run is done on 0.2µg

No special treatment of the protein samples is required, however requires fastidious attention to all steps during sample preparation and LC-MS/MS in order to have the highest reproducibility as possible.

Suggestion:

For these experiments we recommend triplicate analysis of each biological replicate, to increase statistical significance of the peptide (and thus protein) fold-changes measured. This type of experiment can be extended to studies include protein expression as a function of time given an external stimuli or gene knockout, for instance.

Features:

Rapid and low-cost: Label-free methods for both relative and absolute quantitation have been developed as a rapid and low-cost alternative to other quantitative proteomic approaches.

Less reliable for measuring small changes: These strategies are ideal for large-sample analyses in clinical screening or biomarker discovery experiments, and while they are good at measuring large changes in protein expression (> 2 orders of magnitude), they are less reliable for measuring small changes .

Need to be more carefully controlled: Unlike other quantitation methods, label-free samples are separately collected, prepared and analyzed by LC-MS or LC-MS/MS. Because of this, label-free quantitation experiments need to be more carefully controlled than stable isotope methods to account for any experimental variations. Protein quantitation is performed using either ion peak intensity or spectral counting.

Rely on LC-MS only (no MS/MS) when by ion peak intensity, sensitive computer algorithms are required: Relative quantitation by ion peak intensity relies on LC-MS only (no MS/MS). The direct MS m/z values for all ions are detected and their signal intensities at a particular time recorded. The signal intensity from electrospray ionization has been reported to highly correlate with ion concentration, and therefore the relative peptide levels between samples can be determined directly from these peak intensities. Because of the large amount of data collected from these experiments, sensitive computer algorithms are required for automated ion peak alignment and comparison.

Significant normalization is required when by spectral counts: Label-free relative quantitation by spectral counts entails comparing the sum of the MS/MS spectra from a given peptide across multiple samples, which has been shown to directly correlate with protein abundance. Unlike quantitation by peak intensity, spectral counting does not require special algorithms or other tools, although significant normalization is required.

A reference below for your information:

Matthias Gstaiger, Ruedi Aebersold. Applying mass spectrometry-based proteomics to genetics, genomics and network biology [J].Nature Reviews Genetics, 2009, 10:617-627

Figure1 Comparison among MS profiling approaches

Isotope-labeling approaches

As shown in part a in the figure, differential labeling of proteins or peptides with heavy or light isotopes (indicated in red or blue) can be done in vitro or by the incorporation of isotope-labeled amino acids by metabolic labeling in vivo. For in vitro labeling, wild-type (wt) and mutant (mut) samples are prepared separately and isolated proteins or peptides are differentially labeled with heavy or light versions of isotope-tagging reagents, mainly through their sulphhydryl (for example, isotope-coded affinity tags)⁷¹ or amine groups (for example, isotope-coded protein labels)⁷³. Differential labeling introduces a characteristic mass shift, which can be used to determine the MS1 peptide ratios between pairs of heavy and light peptides. Peptide labeling with recently introduced isobaric tags for relative and absolute quantitation, which as the name indicates, keep the mass of the differentially labeled precursor ions of a given peptide constant but allow quantification after tandem mass spectrometry (MS/MS) analysis on the basis of sample-specific reporter ion intensities from up to eight different samples in a single liquid chromatography–tandem mass spectrometry (LC–MS/MS) experiment.

The use of synthetic isotope-labeled reference peptides for absolute quantification that was pioneered by Desiderio et al.⁷⁶ has been extended to proteomic studies by Steve Gygi and colleagues⁷⁷. In this approach, known amounts of synthetic isotope-labeled reference peptides, which correspond to proteotypic peptides of the proteins to be analyzed, are added to the samples before LC–MS/MS analysis for absolute quantification of proteins.

Stable isotope labeling with amino acids in cell culture is an in vivo isotope labeling method that is becoming increasingly popular⁷⁸. Wild-type and mutant cells are grown in media that contains either light or heavy isotope versions of lysine or arginine, which yield differentially labeled proteomes. The entire labeling process occurs at the beginning of the experiment, which has the advantage that samples can be combined at early steps to avoid errors that can be introduced when samples are separately processed. As the method is limited to cells or organisms that can be metabolically labeled, it is not generally applicable to human tissues and body fluids.

Label-free quantification from aligned MS1 spectra

For label-free quantification (part b in the figure) wild-type and mutant proteomes are analyzed by separate LC–MS/MS experiments and the MS1 spectra are computationally aligned to calculate the relative protein abundance changes on the basis of the signal intensities of extracted ion chromatograms from aligned peptide features. This reduces the undersampling problem that is known to occur with MS/MS-based approaches and results in a dynamic range of three to four orders of magnitude⁷⁹. Newer hybrid MS instruments (LTQ FT and LTQ Orbitrap) offer the option to simultaneously record MS signal intensities and identify peptides using MS/MS. These two types of information can be combined by recently developed computational approaches^80,81,82. The number of peptides that can be mapped across different LC–MS/MS experiments therefore depends on the accuracy of the peptide masses that are determined by the mass analyzer and reproducibility of the LC system. Strategies for signal normalization and for correcting variations in LC performances have been developed and are now integrated in automated computational platforms for label-free MS analysis⁸³.

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