Preparative Electrophoresis for HDL Particle Size Separation and Intact-Mass Apolipoprotein Proteoform Analysis.

The most abundant proteins on high-density lipoproteins (HDLs), apolipoproteins A-I (APOA1) and A-II (APOA2), are determinants of HDL function with 15 and 9 proteoforms (chemical-structure variants), respectively. The relative abundance of these proteoforms in human serum is associated with HDL cholesterol efflux capacity, and cholesterol content. However, the association between proteoform concentrations and HDL size is unknown. We employed a novel native-gel electrophoresis technique, clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE) paired with mass spectrometry of intact proteins to investigate this association. Pooled serum was fractionated using acrylamide gels of lengths 8 and 25 cm. Western blotting determined molecular diameter and intact-mass spectrometry determined proteoform profiles of each fraction. The 8- and 25 cm experiments generated 19 and 36 differently sized HDL fractions, respectively. The proteoform distribution varied across size. Fatty-acylated APOA1 proteoforms were associated with larger HDL sizes (Pearson's R = 0.94, p = 4 × 10-7) and were approximately four times more abundant in particles larger than 9.6 nm than in total serum; HDL-unbound APOA1 was acylation-free and contained the pro-peptide proAPOA1. APOA2 proteoform abundance was similar across HDL sizes. Our results establish CN-GELFrEE as an effective lipid-particle separation technique and suggest that acylated proteoforms of APOA1 are associated with larger HDL particles.

Mapping the Proteoform Landscape of Five Human Tissues.

A functional understanding of the human body requires structure-function studies of proteins at scale. The chemical structure of proteins is controlled at the transcriptional, translational, and post-translational levels, creating a variety of products with modulated functions within the cell. The term "proteoform" encapsulates this complexity at the level of chemical composition. Comprehensive mapping of the proteoform landscape in human tissues necessitates analytical techniques with increased sensitivity and depth of coverage. Here, we took a top-down proteomics approach, combining data generated using capillary zone electrophoresis (CZE) and nanoflow reversed-phase liquid chromatography (RPLC) hyphenated to mass spectrometry to identify and characterize proteoforms from the human lungs, heart, spleen, small intestine, and kidneys. CZE and RPLC provided complementary post-translational modification and proteoform selectivity, thereby enhancing the overall proteome coverage when used in combination. Of the 11,466 proteoforms identified in this study, 7373 (64%) were not reported previously. Large differences in the protein and proteoform level were readily quantified, with initial inferences about proteoform biology operative in the analyzed organs. Differential proteoform regulation of defensins, glutathione transferases, and sarcomeric proteins across tissues generate hypotheses about how they function and are regulated in human health and disease.

Next-Generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.

Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS , a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multi-parametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We apply Ig-MS to plasma from subjects with severe & mild COVID-19, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.

New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry.

Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values (R2 = 0.6-0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C (R = 0.5) and glycated ApoA-I with higher fasting glucose (R = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single N-acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference (R ∼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes.