Nonisotopic Reagents for a Cost-Effective Increase in Sample Throughput of Targeted Quantitative Proteomics

Abstract

The new technology of ultrathroughput MS (uMS) transforms the intrinsic capability of analyte multiplexing in mass spectrometry (MS) to sample multiplexing. Core technological advantages of uMS rely on the decoupled use of isotopic quantitation reference and nonisotopic mass coding of samples. These advantages include: (1) high sample-throughput potential, (2) utilization of minimal amounts of expensive stable isotopes for the quantitation reference, and (3) unleashing of the open-source exploration of the chemical structure diversity of nonisotopic reagents to significantly enhance the MS detectability of analytes. A particular uMS method, ultrathroughput multiple reaction monitoring (uMRM), is reported for one-experiment quantitation of a surrogate peptide (SVILLGR) of prostate specific antigen (PSA) in multiple serum samples. Following derivatization of the pair of spiked, isotopic reference (SVILLGR*) and endogenous, native peptide in each sample, all samples were pooled for a step of simultaneous enrichment and cleanup of derivatized peptide pairs using immobilized antibody. The MS analysis of the pooled sample reported the quantity and sample origin of the surrogate peptide. Several analyses with different sample throughput were presented, with the highest being 15-in-1. Screening of nonisotopic reagents used combinatorial libraries of peptidyl compounds, and the reagent selection was based on the derivatization effectiveness and the capability of MS signal enhancement for the peptide. The precision, accuracy, and linearity of the uMRM MS technology were found to be comparable with standard isotope dilution MRM MS.

Determining Linear Free Energy Relationships in Peptide Fragmentation Using Derivatization and Targeted Mass Spectrometry

“…Each resulting peptide was derivatized with a designer aliphatic residue (G, A, V, L, or I) at the N-terminus. The derivatization efficiency was quantified as the percentage of chemical conversion (PCC) of the model peptide SVILLGR, as previously described . An isotopic counterpart peptide, SVIL­[L– 13 C 6 15 N]­GR (SVI*), with a known concentration was used as the quantitation reference.…”

“…The Fmoc-AA-OSu reagents expanded the collection of nonisotopic reagents for multiplexing samples in a single analysis using ultrathroughput MS (uMS) (Text S3). The commercial availability of Fmoc-AA-OSu reagents makes them immediately adaptable for implementing uMS workflows similar to that of uMRM MS. , …”

“…It is problematic to use isotopic reagents − for the investigation of the effects of N-terminal basicity on peptide MS/MS, because of the limited choices of structures and available information on the physical properties of the reagents. In contrast, the separate utilization of isotopic quantitation references and nonisotopic derivatizing reagents, as demonstrated by the method of ultrathroughput multiple-reaction-monitoring (uMRM) MS, , readily exploits unlimited chemical structures for derivatizing peptides. Therefore, the derivatization of peptides with nonisotopic reagents can be rationally designed for the systematic study of N-terminal effects.…”

HST-MRM-MS: A Novel High-Sample-Throughput Multiple Reaction Monitoring Mass Spectrometric Method for Multiplex Absolute Quantitation of Hepatocellular Carcinoma Serum Biomarker

“…However, one of the main challenges that has restricted the application of general MRM-MS analysis is the low sample-throughput, which failed to proceed with efficient quantification and verification of biomarkers due to large numbers of biological samples . Therefore, high sample-throughput MRM-MS techniques are in urgent need.…”

“…In our previous work, we combined mTRAQ for absolute quantification and iTRAQ for relative quantification and have increased the throughput of MRM-MS to four samples in a single run . Recently, Yao and co-workers developed a new MRM-MS approach using nonisotopic reagent labeling that has expanded the throughput up to 15 samples . However, in this multiplexing experiment, target peptides were labeled by 15 nonisotopic reagents with different peaks in a chromatogram.…”