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The ability to quantitatively probe diverse panels of proteins and their post-translational modifications (PTMs) across multiple samples would aid a broad spectrum of biological, biochemical and pharmacological studies. We report a novel, microarray analytical technology that combines immuno-affinity capture with Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI MS), which is capable of supporting highly multiplexed, targeted proteomic assays. Termed "Affinity-Bead Assisted Mass Spectrometry" (Affi-BAMS), this LC-free technology enables development of highly specific and customizable assay panels for simultaneous profiling of multiple proteins and PTMs. While affinity beads have been used previously in combination with MS, the Affi-BAMS workflow uses enrichment on a single bead that contains one type of antibody, generally capturing a single analyte (protein or PTM) while having enough binding capacity to enable quantification within approximately 3 orders of magnitude. The multiplexing capability is achieved by combining Affi-BAMS beads with different protein specificities. To enable screening of bead-captured analytes by MS, we further developed a novel method of performing spatially localized elution of targets from individual beads arrayed on a microscope slide. The resulting arrays of micro spots contain highly concentrated analytes localized within 0.5 mm diameter spots that can be directly measured using MALDI MS. While both intact proteins and protein fragments can be monitored by Affi-BAMS, we initially focused on applying this technology for bottom-up proteomics to enable screening of hundreds of samples per day by combining the robust magnetic bead-based workflow with the high throughput nature of MALDI MS acquisition. To demonstrate the variety of applications and robustness of Affi-BAMS, several studies are presented that focus on the response of 4EBP1, RPS6, ERK1/ERK2, mTOR, Histone H3 and C-MET to stimuli including rapamycin, H2O2, EPO, SU11274, Staurosporine and Vorinostat.
Pubmed ID: 32188029
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A package of over twenty mass spectrometry-based tools primarily geared toward proteomic data analysis and database mining. It can be run from the command line, but is primarily used through a web browser, and there is a public website that allows anyone to use the software without local installation. Tandem mass spectrometry analysis tools are used for database searching and identification of peptides, including post-translationally modified peptides and cross-linked peptides. Support for isotope and label-free quantification from this type of data is provided. MS-Viewer software allows sharing and displaying of annotated spectra from many different tandem mass spectrometry data analysis packages. Other tools include software for analyzing peptide mass fingerprinting data (MS-Fit); prediction of theoretical fragmentation of peptides (MS-Product); theoretical chemical or enzymatic digestion of proteins (MS-Digest); and theoretical modeling of the isotope distribution of any chemical, including peptides (MS-Isotope). Searches using amino acid sequence can be used to identify homologous peptides in a database (MS-Pattern); the use of the combination of amino acid sequence and masses can be used for homologous peptide and protein identification using MS-Homology. Tandem mass spectrometry peak list files can be filtered for the presence of certain peaks or neutral losses using MS-Filter. Given a list of proteins, MS-Bridge can report all potential cross-linked peptide combinations of a specified mass. Given a precursor peptide mass and information about known amino acid presence, absence, or modifications, MS-Comp can report all amino acid combinations that could lead to the observed mass.
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