Mass Spectrometric Immunoassays in Characterization of Clinically Significant Proteoforms

Abstract

Proteins can exist as multiple proteoforms in vivo, as a result of alternative splicing and single-nucleotide polymorphisms (SNPs), as well as posttranslational processing. To address their clinical significance in a context of diagnostic information, proteoforms require a more in-depth analysis. Mass spectrometric immunoassays (MSIA) have been devised for studying structural diversity in human proteins. MSIA enables protein profiling in a simple and high-throughput manner, by combining the selectivity of targeted immunoassays, with the specificity of mass spectrometric detection. MSIA has been used for qualitative and quantitative analysis of single and multiple proteoforms, distinguishing between normal fluctuations and changes related to clinical conditions. This mini review offers an overview of the development and application of mass spectrometric immunoassays for clinical and population proteomics studies. Provided are examples of some recent developments, and also discussed are the trends and challenges in mass spectrometry-based immunoassays for the next-phase of clinical applications.

Keywords: biomarkers; immunoaffinity; mass spectrometry; posttranslational modifications; top-down analysis.

Figure 1

Schematic representation of the differences between single protein biomarker analysis using (a) conventional immunoassays (total protein concentration is measured); and (b) top-down MS-based immunoassays (protein profile and all proteoform concentrations are measured).

Figure 2

Mass spectrometric immunoassay (MSIA) workflow (a) affinity pipettes derivatization with antibody(ies); (b) introducing affinity pipette in biological sample; (c) protein(s) extraction; (d) rinsing non-specifically bounded substances from the affinity pipette; (e) protein(s) elution with matrix; (f) protein detection using MALDI-TOF MS or ESI MS

Figure 3

Mass spectrometric immunoassay (MSIA) method development, validation and application workflow.

Figure 4

Example mass spectra obtained using MSIA, from different individuals expressing single and multiple SAA polymorphic variants. (a). MALDI-TOF mass spectra obtained from analysis of SAA in human plasma sample, using beta lactoglobulin (BL) as an internal reference standard; (b)–(e). Close-up of SAA from 4 different human plasma samples; (b) SAA 1.1 polymorphic variant (expressed are signals from the native SAA 1.1, as well as two SAA 1.1 proteoforms lacking one (des-R) and two (des-RS) N-terminal amino acids); (c) SAA 1.1/1.2 polymorphic variant (two SAA polymorphic variants are expressed, together with the corresponding truncated proteoforms); (d) SAA 1.1/1.3/2.1 polymorphic variant (three SAA polymorphic variants are expressed) and (e) SAA 1.3 polymorphic variant. Note that beside the originating full-length SAA protein, all samples present with truncated proteoforms; SAA proteoforms in the figure are labeled according to the revised nomenclature for serum amyloid A by the nomenclature committee of the international society of amyloidosis [84].

Figure 5

(a) Mass spectra from serum amyloid A (SAA) standard in different dilutions, and beta lactoglobulin (BL) as an internal reference standard (IRS), obtained using MSIA; (b) generated standard curve from SAA and BL as an IRS.