Mass Spectrometric Immunoassays for Discovery, Screening and Quantification of Clinically Relevant Proteoforms

Abstract

Human proteins can exist as multiple proteoforms with potential diagnostic or prognostic significance. MS top-down approaches are ideally suited for proteoforms identification because there is no prerequisite for a priori knowledge of the specific proteoform. One such top-down approach, termed mass spectrometric immunoassay utilizes antibody-derivatized microcolumns for rapid and contained proteoforms isolation and detection via MALDI-TOF MS. The mass spectrometric immunoassay can also provide quantitative measurement of the proteoforms through inclusion of an internal reference standard into the analytical sample, serving as normalizer for all sample processing and data acquisition steps. Reviewed here are recent developments and results from the application of mass spectrometric immunoassays for discovery of clinical correlations of specific proteoforms for the protein biomarkers RANTES, retinol binding protein, serum amyloid A and apolipoprotein C-III.

Simple and Fast Assay for Apolipoprotein E Phenotyping and Glycotyping: Discovering Isoform-Specific Glycosylation in Plasma and Cerebrospinal Fluid

“…The assay was comprised of two steps: 1) immunocapture of apoE from samples using anti-apoE antibody immobilized within a porous microcolumn inside a pipettor tip, and 2) elution and detection of the captured intact apoE with MALDI-TOF MS. This method has been utilized for the analysis of several other proteins [ 33, 34 ]. The workflow for the assay is shown in Fig.…”

Proteoform Analysis to Fulfill Unmet Clinical Needs and Reach Global Standardization of Protein Measurands in Clinical Chemistry Proteomics

“…However, the goal to fully describe a proteome is only partly met when listing large numbers of protein IDs. It has become clear that detailed structural knowledge on proteoforms has become a crucial aspect in many studies that involve proteome analysis [39][40][41]. The same holds true for the in-depth analysis of the heterogeneous character of protein measurands.…”

Conjugating immunoassays to mass spectrometry: Solutions to contemporary challenges in clinical diagnostics

“…This combination of immunoaffinity capture and MS detection can provide substantial improvements in sensitivity and specificity by exploiting the enrichment capabilities of antibodies, while eliminating issues related to non-specific binding by using direct measurement of unique antigen m/z 's [ 59 ]. MSIA workflows are also applicable to both bottom-up and top-down proteomics analyses, the latter of which can identify novel proteoforms without prior knowledge of the complete amino acid sequence and PTMs, therefore providing more complete sequence coverage [ [60] , [61] , [62] ].…”