Sequential Multiplexed Analyte Quantification Using Peptide Immunoaffinity Enrichment Coupled to Mass Spectrometry

TL;DR: The increasing importance of targeted MS technologies in clinical proteomics and the potential key roles these techniques will have in bridging biomedical discovery and clinical implementation are described.

TL;DR: The variety of applied strategies for all "seven critical factors" in current literature on MS-based protein quantification have been critically reviewed and evaluated and the impact of strategies moving away from traditional protein cleavage isotope dilution mass spectrometry (PC-IDMS) toward approaches that are more dedicated to bioanalysis is predicted.

TL;DR: Recent advances in the method and technology for further enhancing SRM sensitivity are reviewed, and its broad biomedical applications in human bodily fluids, tissue and cell lines are highlighted, plus general perspectives on the potential of achieving both high sensitivity and high sample throughput for large‐scale quantification of hundreds of target proteins are discussed.

TL;DR: This study establishes that LC-MRM-MS using stable isotope dilution, with appropriate attention to analytical validation and appropriate quality control measures, enables sensitive, specific, reproducible, and quantitative measurements of proteins and peptides in complex biological matrices such as plasma.

TL;DR: The AQUA strategy was used to quantify low abundance yeast proteins involved in gene silencing, quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay.

TL;DR: This tutorial explains the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions, and normalization and various factors affecting sensitivity and accuracy are discussed.

TL;DR: Anti-peptide antibody enrichment will contribute to increased sensitivity of MS-based assays, particularly for lower abundance proteins in plasma, and may ultimately allow substitution of a rapid bind/elute process for the time-consuming reverse phase separation now used as a prelude to online MS peptide assays.

TL;DR: The ability to measure the abundance of many proteins precisely and simultaneously in experimental samples is an important, recent advance for static and dynamic, as well as descriptive and predictive, biological research.