Title

Methods for peptide and protein quantitation by liquid chromatography-multiple reaction monitoring mass spectrometry

Document Type

Article

Date of Publication

6-1-2011

Publication Title

Molecular and Cellular Proteomics

First Page

M110.006593-1

Last Page

M110.006593-17

Abstract

Abstract

Liquid chromatography-multiple reaction monitoring mass spectrometry of peptides using stable isotope dilution (SID) provides a powerful tool for targeted protein quantitation. However, the high cost of labeled peptide standards for SID poses an obstacle to multiple reaction monitoring studies. We compared SID to a labeled reference peptide (LRP) method, which uses a single labeled peptide as a reference standard for all measured peptides, and a label-free (LF) approach, in which quantitation is based on analysis of un-normalized peak areas for detected MRM transitions. We analyzed peptides from the Escherichia coli proteins alkaline phosphatase and β-galactosidase spiked into lysates from human colon adenocarcinoma RKO cells. We also analyzed liquid chromatography-multiple reaction monitoring mass spectrometry data from a recently published interlaboratory study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer network (Addona et al. (2009) Nat. Biotechnol. 27: 633–641), in which unlabeled and isotopically labeled synthetic peptides or their corresponding proteins were spiked into human plasma. SID displayed the highest correlation coefficients and lowest coefficient of variation in regression analyses of both peptide and protein spike studies. In protein spike experiments, median coefficient of variation values were about 10% for SID and 20–30% for LRP and LF methods. Power calculations indicated that differences in measurement error between the methods have much less impact on measured protein expression differences than biological variation. All three methods detected significant (p < 0.05) differential expression of three endogenous proteins in a test set of 10 pairs of human lung tumor and control tissues. Further, the LRP and LF methods both detected significant differences (p < 0.05) in levels of seven biomarker candidates between tumors and controls in the same set of lung tissue samples. The data indicate that the LRP and LF methods provide cost-effective alternatives to SID for many quantitative liquid chromatography-multiple reaction monitoring mass spectrometry applications.

A rapidly evolving approach to protein quantitation is the targeted analysis of representative peptides by liquid chromatography-tandem mass spectrometry by multiple reaction monitoring (LC-MRM-MS)1 analysis (1–3). In this approach, peptides are quantified by monitoring several MRM transitions for each peptide with either a triple quadrupole or a quadrupole-ion trap instrument. Stable isotope dilution (SID), in which labeled peptides are used as internal standards is considered the gold standard for rigorous quantitation by LC-MRM-MS (1, 4, 5). In contrast to antibody-based quantitation, where antibody availability and specificity are often limiting, LC-MRM-MS enables configuration of an assay for essentially any protein. In practice, this approach has proven sensitive enough to apply to challenging protein quantitation problems. For example, proteins can be quantified at single-digit copy numbers in cells (6) and in plasma at levels approaching ng/ml (7, 8). With antibody-based enrichment, LC-MRM-MS can achieve even greater sensitivity (9–12).

Despite the power of the method, the use of SID is nevertheless limited practically by the cost of labeled standards, which are expensive (∼$1000 per milligram for labeled peptides of high purity). This issue is particularly important in considering LC-MRM-MS to evaluate candidate biomarkers for disease. Application of biomarker discovery platforms, such as shotgun proteomics or transcriptome profiling can yield hundreds of biomarker candidates. The next phase of analysis, termed “verification,” consists of configuring assays for the candidates and evaluating them in well-defined test cohorts (1). The cost of configuring SID-LC-MRM-MS assays for three representative peptides each for 50 proteins would be approximately $150,000.

An MRM-based approach for targeted protein quantitation with a more limited number of isotopically labeled standards could be particularly useful for biomarker candidate screening, in which the expense of labeled standards presents a real barrier to verification of large numbers of candidates. Although SID should outperform methods that do not employ labeled standards for each analyte, there are insufficient data available to evaluate the performance of alternative techniques or to determine appropriate contexts for their use.

Here we compared SID with two alternative methods. The first is a labeled reference peptide (LRP) method, which employs a single isotopically labeled peptide as the reference peptide for all of the other peptide analytes. The second is a label-free (LF) method that employs no standard and where quantitation is based only on the peak areas extracted from LC-MRM-MS product ion chromatograms. We compared these three approaches with datasets from analyses of defined peptide and protein mixtures on triple quadrupole LC-MS instruments. Test samples included synthetic peptides or their corresponding proteins spiked into a human cell lysate. We also analyzed LC-MRM-MS data from a recent study by the National Cancer Institute Clinical Proteomic Technology Assessment for Cancer (CPTAC) program (3), which analyzed human plasma spiked with peptide and protein standards. Finally, we compared the methods in analysis of several lung cancer biomarker candidate proteins in normal lung and lung-tumor tissues. Our studies document the performance of all three methods with the same datasets. The data establish the performance of the LRP and LF methods and provide a basis to select between all three methods for appropriate applications in quantitative proteomics.

DOI

https://doi.org/10.1074/mcp.M110.006593

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