Standardization approaches in absolute quantitative proteomics with mass spectrometry

Abstract

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.

Benchmarking accuracy and precision of intensity-based absolute quantification of protein abundances inSaccharomyces cerevisiae

“…Among the different available MS technologies, quantitative label-free methods have become increasingly popular, due to their relative ease of use and cost-effectiveness, particularly when they are compared to more expensive and laborious methods, as using isotope-labeled peptides [2]. In quantitative label-free approaches, normalization of the raw data is a critical step in determining protein absolute estimates [3][4][5][6].…”

A reflection on the role of ICP-MS in proteomics: Update and future perspective

“…The distinctive feature of this elemental technique is that the element ionization is independent of the chemical structure of the compound containing it. That is particularly advantageous for quantitative purposes, since there is no need to use standards specific to each analyte, as any compound that contains the target detectable element (any but C, H, N, and O, because ICP plasma works at atmospheric pressure) is useful as quantification standard (e.g., inorganic salts) [2,3].…”

“…Species-independent ICP-MS signal response of any heteroatom present in a protein has been long acknowledged as a great asset for the simple and direct certification of SIL peptide and protein standards [3] required in most MS-based absolute quantitate proteomics (i.e., AQUA). Nonetheless, availability of proper characterized standards is not often feasible, and it is required to synthesize and certify those standards.…”

Quantitative mapping of specific proteins in biological tissues by laser ablation–ICP-MS using exogenous labels: aspects to be considered

“…The great analytical challenge in this area is to combine specificity (biological samples are complex matrices), high sensitivity (the species of interest can be at very low concentration levels) and absolute quantitative information (i.e. the capability to assess not only differential protein expression between two samples, but also protein absolute quantities) [1,2]. Optical-spectroscopy and mass spectrometry (MS) techniques offer interesting capabilities within this field for liquid samples and for chemical imaging of solid specimens [3,4].…”

“…Low detection limits, wide linear dynamic range, elemental specificity, multi-element (and multi-isotope) analysis, and virtual species-and matrix-independent ionization, are among the main analytical features of ICP-MS. Such performances are of particular interest to obtain absolute concentrations of biomolecules in complex samples without the need of specific standards [1,7].…”