Peptide Selection for Targeted Protein Quantitation

Abstract

Targeted proteomics methods in their different flavors rely on the use of a few peptides as proxies for protein quantitation, which need to be specified either prior or after data acquisition.However, in contrast to discovery methods that use all identified peptides for a given protein to estimate its abundance, targeted proteomics methods are limited in the number of peptides that are used for protein quantitation. As only few peptides per protein are acquired or extracted in targeted experiments, the selection of peptides that are used for targeted protein quantitation becomes crucial. Several rules have been proposed to guide peptide selection for targeted proteomics studies, which have generally been based on the amino acidic composition of the peptide sequences. However, the compliance of these rules do not imply that not-conform peptides are not reproducibly generated nor they guarantee that the selected peptides correctly represent the behaviour of the protein abundance in different conditions.

What is targeted proteomics? A concise revision of targeted acquisition and targeted data analysis in mass spectrometry

“…Typically, three to five fragment ions per peptide are measured, and one to three unique peptides with good quantitative properties are used for protein quantification (Fig. B) . The time between two sampling events of the same transition is the cycle time , and the time that the mass spectrometer dedicates to each transition is the dwell time .…”

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia

“…In contrast, the targeted analysis of peptides by PRM is recognised to result in a more reproducible quantification but highly dependent on a correct selection of proteotypic peptides being targeted for quantiation . Although some rules have been proposed to guide the peptide selection, there is no guarantee that the selected peptides are not prone to undergo chemical or enzymatic modifications or is found with missed or nonsense cleavages, which naturally will impact the accuracy of the quantification . Ideally, orthogonal non‐MS‐based methods (e.g., ELISA, immunoblot, immunohistochemistry) are required in order to validate the protein expression patterns in an independent cohort of samples.…”

“…[26] Although some rules have been proposed to guide the peptide selection, there is no guarantee that the selected peptides are not prone to undergo chemical or enzymatic modifications or is found with missed or nonsense cleavages, which naturally will impact the accuracy of the quantification. [27] Ideally, orthogonal non-MS-based methods (e.g., ELISA, immunoblot, immunohistochemistry) are required in order to validate the protein expression patterns in an independent cohort of samples. Recent reviews summarized the proteomics studies aiming to find tissues potential biomarkers for PCa diagnosis or prognosis.…”

Performing Quantitative Determination of Low-Abundant Proteins by Targeted Mass Spectrometry Liquid Chromatography

“…Peptides used for LC-MS/MS identification usually contains between 6 and 20 amino acids in sequence [37]. The proteotypic peptides should ideally not contain amino acids which are prone to modifications, either during sample preparation or in the biological system.…”