PSAQ™ standards for accurate MS–based quantification of proteins: from the concept to biomedical applications

Abstract

Absolute protein quantification, i.e. determining protein concentrations in biological samples, is essential to our understanding of biological and physiopathological phenomena. Protein quantification methods based on the use of antibodies are very effective and widely used. However, over the last ten years, absolute protein quantification by mass spectrometry has attracted considerable interest, particularly for the study of systems biology and as part of biomarker development. This interest is mainly linked to the high multiplexing capacity of MS analysis, and to the availability of stable-isotope-labelled standards for quantification. This article describes the details of how to produce, control the quality and use a specific type of standard: Protein Standard Absolute Quantification (PSAQ™) standards. These standards are whole isotopically labelled proteins, analogues of the proteins to be assayed. PSAQ standards can be added early during sample treatment, thus they can correct for protein losses during sample prefractionation and for incomplete sample digestion. Because of this, quantification of target proteins is very accurate and precise using these standards. To illustrate the advantages of the PSAQ method, and to contribute to the increase in its use, selected applications in the biomedical field are detailed here.

Differential secretome analysis of Pseudomonas syringae pv tomato using gel-free MS proteomics

“…PSAQ offers a number of advantages compared to other internal standard strategies, such as AQUA or Qconcat, due to the biochemical identity of the labeled full-length protein with that of the analyte protein. Both protein species are subject to identical sampling handling such that any decrease in output signal, for instance as a result of incomplete trypsin digestion, is common to both and thus controlled for internally (Picard et al, 2012). Purities of labeled HrpA1 and AvrPto1 were determined to be 96 and 52% (Supplementary Figure 1) by relative band fluorescence intensities of SYPRO ® Ruby stained SDS-PAGE gels.…”

The complexity of protein semiochemistry in mammals

“…A subtle variant of the QconCAT approach is provided by the PCS (peptide-concatenated standard) strategy, in which peptides are interspersed with hexapeptide sequences that recapitulate the native primary sequence context, theoretically balancing the rate of proteolysis of analyte and standard [44]. An even more comprehensive approach is provided by PSAQ (protein standards for absolute quantification) in which an entire protein is expressed and labelled in recombinant form to generate multiple tryptic peptides for quantification [28,[45][46][47]. However, expression of MUPs as PSAQ standards would also generate a large number of common or shared peptides.…”

Psoriatic Arthritis Under a Proteomic Spotlight: Application of Novel Technologies to Advance Diagnosis and Management

“…Keshishian et al [115] Collins et al integrated 48 putative biomarkers of drug toxicity from diverse data sets into a MRM assay. The assay was deployed to assess the levels of these biomarkers in the liver tissue of rats that were treated with the hepatoxic compound EMD 335823.…”

“…As a targeted peptide-centric approach, MRM has been used for multiple biomarker studies in the past few years to detect and quantify biomarkers in a range of diseases (Table 1) [112][113][114][115][116][117][118][119][120][121][122][123]. MRM assays can be multiplexed to support the measurement and quantification of 100 s of important candidate biomarker proteins simultaneously in several hundreds of patient tissue or serum samples.…”