Development and applications of novel mass spectrometry-based proteomic tools and strategies

Jacob Anthony Galan, Purdue University

Abstract

Mass spectrometry-based proteomics has emerged as a powerful approach to study various biological processes, post-translational modifications, disease biomarker detection, or elucidation of complex sample composition. However, such an approach relies heavily on the ability of chemical and biochemical reagents to aid in protein quantification and enrichment of specific target proteins from complex samples. In this work, to address the issues described above, we present the design and synthesis of two effective labeling reagents: soluble polymer-based isotope labeling (SoPIL) and isotope tag on amino groups (iTAG) for mass spectrometry-based quantification. We also describe robust proteomic strategies to screen and identify binding proteins and substrates of Syk kinase by using tandem SH2 pulldown and GFP nanotrap purification. The SoPIL reagent (Chapter 3) was introduced and developed to selectively capture cysteine-containing peptides for quantification, which are then released with an isotope tag and analyzed by mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting and fibrinogenolytic activities of crude venoms were measured and correlated with differences in protein abundance determined by the SoPIL analysis. The SoPIL approach is capable of providing an efficient and widely applicable tool for quantitative proteomics. We introduced another isotopic labeling reagent, iTAG (Chapter 4 and 5), as an amine labeling reagent to be used at both protein and peptide levels. The iTAG reagents were prepared with simple, economical procedures and characterized with the measurement of peptides in model peptide mixtures and in complex samples. For protein level labeling, we used iTAG to study early-stage Metabolic Syndrome (MetS) in the Ossabaw swine large animal model. Analysis of Ossabaw swine plasma identified and quantified over 130 proteins. These quantified proteins included up-regulated Apoliporotein A-1, A-II, A-IV, B, E, and ovarian and testicular apolipoprotein N. For peptide level labeling, we coupled iTAG with anti-GFP single-chain antibody to study Syk location-specific associations with multiple proteins. We demonstrate that the single-chain VHH (GFP nanotrap) allowed us to identify interacting partners of the Syk kinase bearing a GFP epitope tag with high efficiency and high specificity. Interacting proteins identified included CrkL, BLNK, α and β-tubulin, and Csk. While the results revealed that the new quantitative proteomic strategy is generally applicable for integration of protein interaction data with subcellular localization, extra caution should be considered while evaluating the results obtained by such affinity purification strategies as many false positive interactions can occur after cell lysis. We further expanded the proteomic approach to employ the GST tagged Syk tandem SH2 domain purification to identify Syk- binding proteins and substrates (Chapter 6). Among identified proteins, we further verified Nek9, a serine/threonine kinase with little known function, as a Syk-interacting protein. Inhibition of Syk with piceatannol, a Syk inhibitor, or the use of DT40 Syk-negative chicken B cells resulted in the loss of Nek9 tyrosine phosphorylation. Syk-induced tyrosine phosphorylation of Nek9 was further confirmed using an in vitro kinase assay resulting in increased Nek9 activity, and phosphorylation on tyrosines 520 and 521. Our results suggest that Syk could be the activating kinase for Nek9 in BCR signaling, leading to further downstream signaling cascades. In conclusion, development and applications of novel mass spectrometry-based proteomic tools and strategies provide robust and quantifiable proteomic analyses for diverse biological settings.

Degree

Ph.D.

Advisors

Geahlen, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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