Top-Down Mass Spectrometry for Trace Level Quantification of Staphylococcal Enterotoxin A Variants

Abstract

We addressed here the need for improved sensitivity of top-down mass spectrometry for identification, differentiation, and absolute quantification of sequence variants of SEA, a bacterial toxin produced by Staphylococcus aureus and regularly involved in food poisoning outbreaks (FPO). We combined immunoaffinity enrichment, a protein internal standard, and optimized acquisition conditions, either by full-scan high-resolution mass spectrometry (HRMS) or multiplex parallel reaction monitoring (PRM) mode. Deconvolution of full-scan HRMS signal and PRM detection of variant-specific fragment ions allowed confident identification of each SEA variant. Summing the PRM signal of variant-common fragment ions was most efficient for absolute quantification, illustrated by a sensitivity down to 2.5 ng/mL and an assay variability below 15%. Additionally, we showed that relative PRM fragment ion abundances constituted a supplementary specificity criterion in top-down quantification. The top-down method was successfully evaluated on a panel of enterotoxin-producing strains isolated during FPO, in parallel to the conventional whole genome sequencing, ELISA, and bottom-up mass spectrometry methods. Top-down provided at the same time correct identification of the SEA variants produced and precise determination of the toxin level. The raw files generated in this study can be found on PASSEL (Peptide Atlas) under data set identifier PASS01710.

Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach

“…Lefebvre et al established their quantification method of enterotoxin (SE) variants in vitro by incorporating both internal and external calibration standards. 26 They relied on peak areas from either the extracted ion chromatogram (XIC) or deconvoluted mass peak quantification. Although it is an interesting approach for trace-level proteins, it remains too specific for these kinds of proteins and not applicable for routine albumin analysis.…”

“…Only recently has the top-down LC-MS method for absolute quantification based on deconvolution started emerging. Lefebvre et al established their quantification method of enterotoxin (SE) variants in vitro by incorporating both internal and external calibration standards . They relied on peak areas from either the extracted ion chromatogram (XIC) or deconvoluted mass peak quantification.…”

“…Conventional antigen–antibody-based methods, such as immunoturbidimetry, can directly quantify a protein by measuring the light scattered by the immune complexes formed but are unable to distinguish isoforms. , Proteomic approaches based on mass spectrometry were investigated as a possible solution to this limitation. Bottom-up strategies, a peptide-centric proteomic approach, offer deep proteome coverage and reliable quantification of isoforms, but it is unsuitable for clinical settings due to time-consuming sample preparation procedures, data processing, and loss of information related to the 3D structure caused by proteolytic digestion. ,,, Since the introduction of nebulization–ionization technologies in top-down strategies, liquid chromatography–high-resolution mass spectrometry (LC-HR-MS) techniques gained popularity for native-like folded-state analyses of molecules, ,,− specifically for isoform characterization. , In this way, we recently reported the detection and quantification of several HSA isoforms with minimal sample preparation and relatively short acquisition time. , …”

FLASHQuant: a fast algorithm for proteoform quantification in top-down proteomics

“…LFQ of proteoforms in most cases still relies on deconvolution-centric software, such as Protein Deconvolution (Thermo Fisher Scientific) 22,23 , DataAnalysis (Bruker Daltonics) 24–26 , TopPIC suite 6,27,28 , and ProMex 29,30 . As the primary goal of deconvolution tools is to detect proteoforms’ masses rather than quantify them, chromatographic information needed for quantification is widely neglected on these platforms.…”

“…Existing LFQ data analysis tools for MS1 level quantification (including chromatographic feature detection and intensity calculation) still need further developments and improvements regarding reproducibility, overlapping signal resolution, or usability. LFQ of proteoforms in most cases still relies on deconvolution-centric software, such as Protein Deconvolution (Thermo Fisher Scientific) 22,23 , DataAnalysis (Bruker Daltonics) [24][25][26] , TopPIC suite 6,27,28 , and ProMex 29,30 . As the primary goal of deconvolution tools is to detect proteoforms' masses rather than quantify them, chromatographic information needed for quantification is widely neglected on these platforms.…”