Methods for Peptide and Protein Quantitation by Liquid Chromatography-Multiple Reaction Monitoring Mass Spectrometry

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 ␤-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 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 singledigit 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).

Chronic Nicotine Treatment Impacts the Regulation of Opioid and Non-opioid Peptides in the Rat Dorsal Striatum

“…To cope with this issue, we selected an internal standard of similar hydrophobicity to each targeted endogenous peptide, which also has close retention time on the C18 column (29), to correct the variations of its content caused during the LC-MS data acquisition, in particular during sample treatment. Compared with the label-free method, previous studies have shown that the use of even a single labeled peptide as a reference standard allows for the increased precision for measuring all other measured endogenous peptides (30). Consistent with this conclusion, another study also showed that the use of synthetic heavy peptides as analog or isotopically labeled internal standards allows the improvement in precision and accuracy of peptide analysis (21).…”

Assembly Dynamics and Stoichiometry of the Apoptosis Signal-regulating Kinase (ASK) Signalosome in Response to Electrophile Stress

“…The LRP method requires only a single labeled reference standard and thus offers the most cost-effective approach to a targeted survey of 90 candidate binding partners. However, the method is potentially subject to ionization suppression and interferences because of the lack of labeled standards for all analytes (29). Thus, our use of SID-PRM analyses of a single peptide for each of 26 candidate ASK1-interacting proteins enabled highly precise quantitation, which was sufficient for estimates of complex stoichiometries.…”

“…This was followed by 14 targeted MS2 scans at a resolution of 17,500 and with an AGC value of 5e5, a max injection time of 80 msec, a 0.5 m/z isolation window, a fixed first mass of 150 m/z, normalized collision energy of 27, and recorded as profile data. The targeted-MS2 methods were controlled with a timed inclusion list containing the target precursor m/z value, charge, and a 3 min retention time window that was determined from prior analyses of synthetic peptide standards as described (29). Lists of all peptides targeted for PRM analysis are given in supplemental Tables S1-S3.…”

“…The first relied on normalization of each unlabeled target peptide to a heavy isotope labeled peptide standard called the labeled reference peptide (LRP) for relative quantitation (29). Three LRP standards (GYSFTTTAERˆ, AAQGDITAPGGARˆ, and APLDNDIGVSEATRˆ) were purchased from New England Peptide (Gardner, MA) at Ͼ98% purity and quantified by amino acid analysis.…”

Quantitative Profiling of Protein Tyrosine Kinases in Human Cancer Cell Lines by Multiplexed Parallel Reaction Monitoring Assays

“…Instrument quality control assessment was done as described previously (24). Quantitative analyses were done by the labeled reference peptide (LRP) method we described previously (24) using U-13 C 6 , U-15 N 4 -arginine-labeled alkaline phosphatase (AP) peptide (AAQGDITAPGGA*R), ␤-galactosidase (BG) peptide (APLDNDIGVSEAT*R), and ␤-actin (ACTB) peptide (GYSFTTTAE*R) as the reference standard mixture (New England Peptide, Gardner, MA).…”