Quantitative mass spectrometry in proteomics: a critical review

Abstract

The quantification of differences between two or more physiological states of a biological system is among the most important but also most challenging technical tasks in proteomics. In addition to the classical methods of differential protein gel or blot staining by dyes and fluorophores, mass-spectrometry-based quantification methods have gained increasing popularity over the past five years. Most of these methods employ differential stable isotope labeling to create a specific mass tag that can be recognized by a mass spectrometer and at the same time provide the basis for quantification. These mass tags can be introduced into proteins or peptides (i) metabolically, (ii) by chemical means, (iii) enzymatically, or (iv) provided by spiked synthetic peptide standards. In contrast, label-free quantification approaches aim to correlate the mass spectrometric signal of intact proteolytic peptides or the number of peptide sequencing events with the relative or absolute protein quantity directly. In this review, we critically examine the more commonly used quantitative mass spectrometry methods for their individual merits and discuss challenges in arriving at meaningful interpretations of quantitative proteomic data.

Analytical strategies for phosphoproteomics

“…The various quantitation strategies are outside the scope of this review. They are described in more details in other recent reviews e.g., by Bantscheff and co-workers [131].…”

pH‐regulated formation of side products in the reductive amination approach for differential labeling of peptides in relative quantitative experiments

“…The stable isotope labeling techniques developed in the last two decades for relative quantitation experiments space from in vivo metabolic incorporation of heavy isotopes, or isotopically [3][4][5][6]. Many recent reviews give a comprehensive picture of the current tendency for relative quantitative proteomics with stable isotope labeling [7][8][9][10][11].…”

“…The stable isotope labeling techniques developed in the last two decades for relative quantitation experiments space from in vivo metabolic incorporation of heavy isotopes, or isotopically [3][4][5][6]. Many recent reviews give a comprehensive picture of the current tendency for relative quantitative proteomics with stable isotope labeling [7][8][9][10][11].Reductive amination with formaldehyde and NaCNBH 3 is a simple reaction that involves all the free amino groups of peptides, namely N-termini and lysine residues, replacing hydrogens with two methyl groups [12]. A differential isotope labeling can be achieved by employing d(0)-formaldehyde and d(2)-formaldehyde obtaining a mass increment of 28 and 32 Da, respectively, for each derivatized reactive site.…”

Challenges in cancer research and multifaceted approaches for cancer biomarker quest

“…Development of hybrid methods coupling MRM assay with protein enrichment by immuno-depletion or enrichment of the peptides by antibody capture and further improvements may be capable of extending MRM method to full dynamic range of plasma proteins. With this view in mind, MRM measurement of plasma peptide may provide a rapid and specific assay platform for high throughput biomarker validation [55,56].…”