Ryan Bomgarden

Carbonyl-Reactive Tandem Mass Tags for the Proteome-Wide Quantification of N-Linked Glycans

N-Linked protein glycosylation is one of the most prevalent post-translational modifications and is involved in essential cellular functions such as cell-cell interactions and cellular recognition as well as in chronic diseases. In this study, we explored stable isotope labeled carbonyl-reactive tandem mass tags (glyco-TMTs) as a novel approach for the quantification of N-linked glycans. Glyco-TMTs bearing hydrazide- and aminooxy-functionalized groups were compared for glycan reducing end derivatization efficiency and quantification merits. Aminooxy TMTs outperform the hydrazide reagents in terms of labeling efficiency (>95% vs 65% at 0.1 μM) and mass spectrometry based quantification using heavy/light-TMT labeled glycans enabled accurate quantification in MS1 spectra (CV < 15%) over a broad dynamic range (up to 1:40). In contrast, isobaric TMT labeling with quantification of reporter ions in tandem mass spectra suffered from severe ratio compression already at low sample ratios. To demonstrate the practical utility of the developed approach, we characterized the global N-linked glycosylation profiles of the isogenic human colon carcinoma cell lines SW480 (primary tumor) and SW620 (metastatic tumor). The data revealed significant down-regulation of high-mannose glycans in the metastatic cell line.

Proteomic quantification and site-mapping of S-nitrosylated proteins using isobaric iodoTMT reagents.

S-Nitrosylation is a redox-based protein post-translational modification in response to nitric oxide signaling and is involved in a wide range of biological processes. Detection and quantification of protein S-nitrosylation have been challenging tasks due to instability and low abundance of the modification. Many studies have used mass spectrometry (MS)-based methods with different thiol-reactive reagents to label and identify proteins with S-nitrosylated cysteine (SNO-Cys). In this study, we developed a novel iodoTMT switch assay (ISA) using an isobaric set of thiol-reactive iodoTMTsixplex reagents to specifically detect and quantify protein S-nitrosylation. Irreversible labeling of SNO-Cys with the iodoTMTsixplex reagents enables immune-affinity detection of S-nitrosylated proteins, enrichment of iodoTMT-labeled peptides by anti-TMT resin, and importantly, unambiguous modification site-mapping and multiplex quantification by liquid chromatography–tandem MS. Additionally, we significantly improved anti-TMT peptide enrichment efficiency by competitive elution. Using ISA, we identified a set of SNO-Cys sites responding to lipopolysaccharide (LPS) stimulation in murine BV-2 microglial cells and revealed effects of S-allyl cysteine from garlic on LPS-induced protein S-nitrosylation in antioxidative signaling and mitochondrial metabolic pathways. ISA proved to be an effective proteomic approach for quantitative analysis of S-nitrosylation in complex samples and will facilitate the elucidation of molecular mechanisms of nitrosative stress in disease.

Dual Labeling Biotin Switch Assay to Reduce Bias Derived from Different Cysteine Subpopulations: A Method to Maximize S-Nitrosylation Detection

RATIONALE S-nitrosylation (SNO), an oxidative post-translational modification of cysteine residues, responds to changes in the cardiac redox-environment. Classic biotin-switch assay and its derivatives are the most common methods used for detecting SNO. In this approach, the labile SNO group is selectively replaced with a single stable tag. To date, a variety of thiol-reactive tags have been introduced. However, these methods have not produced a consistent data set, which suggests an incomplete capture by a single tag and potentially the presence of different cysteine subpopulations. OBJECTIVE To investigate potential labeling bias in the existing methods with a single tag to detect SNO, explore if there are distinct cysteine subpopulations, and then, develop a strategy to maximize the coverage of SNO proteome. METHODS AND RESULTS We obtained SNO-modified cysteine data sets for wild-type and S-nitrosoglutathione reductase knockout mouse hearts (S-nitrosoglutathione reductase is a negative regulator of S-nitrosoglutathione production) and nitric oxide-induced human embryonic kidney cell using 2 labeling reagents: the cysteine-reactive pyridyldithiol and iodoacetyl based tandem mass tags. Comparison revealed that <30% of the SNO-modified residues were detected by both tags, whereas the remaining SNO sites were only labeled by 1 reagent. Characterization of the 2 distinct subpopulations of SNO residues indicated that pyridyldithiol reagent preferentially labels cysteine residues that are more basic and hydrophobic. On the basis of this observation, we proposed a parallel dual-labeling strategy followed by an optimized proteomics workflow. This enabled the profiling of 493 SNO sites in S-nitrosoglutathione reductase knockout hearts. CONCLUSIONS Using a protocol comprising 2 tags for dual-labeling maximizes overall detection of SNO by reducing the previously unrecognized labeling bias derived from different cysteine subpopulations.

Development and evaluation of a fluorescent antibody-drug conjugate for molecular imaging and targeted therapy of pancreatic cancer

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer.

AminoxyTMT: A novel multi-functional reagent for characterization of protein carbonylation

Protein carbonylation is a common oxidative stress (OS)-driven post-translational modification (PTM). Proteome-wide carbonylation events can best be characterized using a combination of analytical approaches. Immunoblotting of carbonylated proteins provides data on the extent of modifications within complex samples, as well as a broad comparison of carbonylation profiles between different biological states (e.g., disease versus control), while mass spectrometry (MS)-based analysis provides information on proteins susceptible to carbonylation, as well as the potential for quantitative characterization of specific sites of amino acid modification. Here, we present a novel use for aminoxyTMT, a derivative of the Tandem Mass Tag (TMT) isobaric labeling reagent, which utilizes an aminooxy functional group for covalent labeling of reactive carbonyls in proteins. When coupled with anti-TMT antibody, we demonstrate the use of aminoxyTMT for immunoblot profiling of protein carbonylation in complex mixtures, as well as enrichment of modified peptides from these mixtures. Proof-of-principle experiments also show the amenability of aminoxyTMT-labeled carbonylated peptides enriched from complex mixtures to identification using tandem MS (MS/MS) and database searching, as well as quantitative analysis using TMT-based reporter ion intensity measurements.

Breast tumors educate the proteome of stromal tissue in an individualized but coordinated manner

Proteomic analysis of the tumor-associated stroma reveals extensive and coordinated regulation by breast cancers. Profiling the tumor stroma proteome Communication between a tumor and cells in the surrounding stroma contributes to tumor growth, progression, and drug resistance. Thus, targeting this communication, in the primary tumor and especially in metastatic niches, may be an effective way to treat cancer. Wang et al. grew patient breast tumors subcutaneously in mice and obtained species-distinguished proteomic profiles of the tumors (human) and tumor-associated stroma (mouse). The authors found that all breast tumors consistently altered clustered subsets of the stromal proteome, particularly proteins involved in immune signaling, but that these varied in a subtype- and stage-specific manner. These findings may have future implications for treatment stratification and provide a platform from which to understand this experimental model and tumor-stroma interactions on a large-scale protein level. Cancer forms specialized microenvironmental niches that promote local invasion and colonization. Engrafted patient-derived xenografts (PDXs) locally invade and colonize naïve stroma in mice while enabling unambiguous molecular discrimination of human proteins in the tumor from mouse proteins in the microenvironment. To characterize how patient breast tumors form a niche and educate naïve stroma, subcutaneous breast cancer PDXs were globally profiled by species-specific quantitative proteomics. Regulation of PDX stromal proteins by breast tumors was extensive, with 35% of the stromal proteome altered by tumors consistently across different animals and passages. Differentially regulated proteins in the stroma clustered into six signatures, which included both known and previously unappreciated contributors to tumor invasion and colonization. Stromal proteomes were coordinately regulated; however, the sets of proteins altered by each tumor were highly distinct. Integrated analysis of tumor and stromal proteins, a comparison made possible in these xenograft models, indicated that the known hallmarks of cancer contribute pleiotropically to establishing and maintaining the microenvironmental niche of the tumor. Education of the stroma by the tumor is therefore an intrinsic property of breast tumors that is highly individualized, yet proceeds by consistent, nonrandom, and defined tumor-promoting molecular alterations.

Abstract 1837: Quantitative analysis of AKT/mTOR pathway using immunoprecipitation and targeted mass spectrometry

Background: PI3K/AKT/mTOR pathway plays a central role in tumor progression and anti-cancer drug resistance. The quantitative measurement of protein expression and PTM status of AKT/mTOR pathway proteins is necessary for precise characterization of the disease, monitor cancer progression and determine treatment response. A major bottleneck in the quantitation of AKT/mTOR pathway proteins is the lack of rigorously validated methods/reagents and a reliance on semi-quantitative results from Western blotting. Mass Spectrometry (MS) is increasingly becoming the detection methodology of choice for protein abundance and modifications. Immunoprecipitation (IP) is commonly used upstream of MS as an enrichment tool for low-abundant protein targets. In addition to protein identification, IP can be combined with targeted MS to identify proteins of interest and protein-protein interactions. Here, we used optimized IP-MS to enrich multiple AKT/mTOR pathway proteins simultaneously for targeted selected reaction monitoring (SRM)-based quantitation of protein levels in two human carcinoma cell lines. Methods: A549 and HCT116 cells were stimulated with EGF or IGF. Several AKT/mTOR pathway targets were enriched by improved IP using Protein A/G and Streptavidin magnetic beads and IP eluates were processed using in-solution digestion for LC-MS analysis. A targeted SRM MS assay was developed for quantitation of AKT/mTOR pathway target peptides (EGFR, AKT2, AKT1, PTEN, PIK3CA, and PIK3R1). Multiple targets were also immunoprecipitated simultaneously and quantitated by targeted SRM assay. Improved IP combined with targeted MS workflow was applied to assess recovery of recombinant proteins in a human plasma matrix. Results: Immunoprecipitation resulted in overall higher yield of target protein and less non-specific binding compared to unenriched samples. This enabled us to combine multiple target antibodies to simultaneously enrich multiple pathway protein targets. Enrichment of total and phosphorylated forms of EGFR, AKT isoforms, PI3K and PTEN resulted in quantitation of low to sub nanogram levels of targets in two cell lysates by targeted MS (LC-SRM/MS). In addition, IP coupled with targeted MS was used to enrich and quantify as low as 10ng/mL recombinant EGFR, AKT2/AKT1, PTEN and PI3K spiked into a human plasma matrix. Conclusion: IP combined with targeted MS permits absolute quantitation of AKT/mTOR pathway proteins and PTMs at low to sub nanogram concentrations. This multiplex targeted assay can be used for verification and validation of AKT/mTOR pathway proteins in other cancer cell lines or tissue samples. Citation Format: Bhavinkumar Patel, Suzanne Smith, Ryan Bomgarden, Kay Opperman, Barbara Kaboord, John Rogers. Quantitative analysis of AKT/mTOR pathway using immunoprecipitation and targeted mass spectrometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1837. doi:10.1158/1538-7445.AM2015-1837

Sensitive and Accurate Quantitation of Phosphopeptides Using TMT Isobaric Labeling Technique

Phosphorylation is an essential post-translational modification for regulating protein function and cellular signal transduction. Mass spectrometry (MS) combined with isobaric tandem mass tags (TMTs) has become a powerful platform for simultaneous, large-scale phospho-proteome site identification and quantitation. To improve the accuracy of isobaric tag-based quantitation in complex proteomic samples, MS3-based acquisition methods such as Synchronous Precursor Selection (SPS) have been used. However, the method suffers from lower peptide identification rates when applied to enriched phosphopeptide samples compared with unmodified samples due to differences in phosphopeptide fragmentation patterns during tandem MS. We developed and optimized two new acquisition methods for analysis of TMT-labeled multiplexed phosphoproteome samples, which resulted in more phosphopeptide identifications with less ratio distortion when compared with previous methods. We also applied these improved methods to a large-scale study of phosphorylation levels in A549 cell lines treated with insulin or insulin growth factor 1 (IGF-1). Overall, 3378 protein groups and 12 465 phosphopeptides were identified, of which 10 436 were quantified across 10 samples without prefractionation. The accurate measurement enabled us to map to numerous signaling pathways including mechanistic target of rapamycin (mTOR), epidermal growth factor receptor (EGFR, ErbB), and insulin signaling pathways.

Abstract LB-267: The proteomic landscape of patient-derived breast cancer xenografts reveals tumor-specific differences in the breast tumor microenvironment

Crosstalk between the tumor and surrounding microenvironment has emerged as an important regulator of tumor growth, metastasis and drug response. Patient-derived breast xenografts (PDXs) closely mimic the tumor microenvironment including the tumor architecture and interactions among cancer cells and stromal cells. PDXs provide a unique opportunity to study tumor-stroma interactions and the regulation of protein expression in the tumor microenvironment since species-specific amino acid sequences of the tumor (human) can be distinguished from the stroma (mouse) by LC-MS. However, quantitative proteomics workflows usually do not report species-specific peptides. We therefore developed a proteomics workflow based on 10-plex isobaric tagging to quantitatively profile the protein expression of PDXs and their associated microenvironment. Three biological replicates of seven breast cancer PDXs, representing three breast cancer subtypes, were profiled. Selecting only gene- and species-specific peptides for quantification of protein expression, we identified 8,113 human proteins (4,867 genes) and 2,251 mouse proteins (1,763 genes). Surprisingly, hierarchical clustering by mouse protein expression tightly clustered 4 of the 7 PDXs, with each of the 3 biological replicates next to one another. Notably, the 4 tightly clustered PDXs were from tumors with claudin-low, Her2-E and luminal B subtypes, whereas the biological replicates of three basal subtypes in the dataset were not tightly clustered. Gene set enrichment analysis of the stromal protein expression revealed upregulation of MTORC1 signaling, EMT, and interferon gamma response signaling with false discovery rates below 5%. We further investigated expression of signaling proteins in the tumor microenvironment by enriching active kinases with multiplexed kinase inhibitor beads. 152 mouse kinases were identified in the tumor microenvironment many tumor-specific differences in kinase levels. Taken together, our results imply that individual patient-derived breast tumors can actively and consistently orchestrate unique alterations in the proteins expressed in their microenvironment. Furthermore, we demonstrate the utility of our proteomic analysis workflow to delineate tumor-stroma signaling networks in PDXs. Citation Format: Xuya Wang, Petra Erdmann-Gilmore, Rosa Viner, Matthew Meyer, Tim Stuhlmiller, Sherri Davies, Shunqiang Li, Qiang Zhang, Arshag Mooradian, Kuan-lin Huang, Ryan Bomgarden, Li Ding, Matthew Ellis, John Rogers, Gary Johnson, Reid Townsend, David Fenyo, Jason M. Held. The proteomic landscape of patient-derived breast cancer xenografts reveals tumor-specific differences in the breast tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-267.

Abstract 3884: Quantitative analysis of IGF1R/AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry

Background: The quantitative measurement of protein expression and modification status of AKT/mTOR pathway proteins is necessary for precise characterization of the disease, monitoring cancer progression and determining treatment response. A major bottleneck in the quantitation of signaling pathway proteins is the lack of rigorously validated methods/reagents and a reliance on semi-quantitative results from current immunoassay technologies (Western blot, ELISA and Luminex). Mass Spectrometry (MS) is increasingly becoming the detection methodology of choice for proteins and their post-translational modifications (PTMs). Immunoprecipitation (IP) is commonly used upstream of MS as an enrichment tool for low-abundant protein targets. In addition to protein identification, IP can be combined with targeted MS to quantitate proteins of interest and identify protein-protein interactions. The objective of this study was to determine the efficacy of multiplex IP to targeted MS technique for measurement of the total and phosphorylated AKT/mTOR pathway targets and to evaluate whether multiplex IP-MS assays are as effective as the current single-plex immunoassay (WB and ELISA) and multiplex Luminex assays. Methods: Serum starved HCT116, MCF7 and A549 cells were stimulated with IGF-1. Multiplex IP to targeted MS assays (mIP-tMS) were developed and validated for absolute quantitation of eleven total and ten phosphorylated AKT/mTOR pathway targets (IGF1R, IR, IRS1, PTEN, AKT, mTOR, GSK3α, GSK3β, TSC2, p70S6K and PRAS40). Validated mIP-tMS assays were benchmarked against currently available WB, ELISA and multiplex Luminex immunoassays across three unstimulated and IGF-1 stimulated cell lysates. Results: In previous work, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads can increase target protein yield with low non-specific background. In this study, we validated multiple antibodies for eleven total and ten phosphorylated AKT/mTOR pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for all eleven total and ten phosphorylated targets in low to sub nanogram concentrations across three unstimulated and IGF-1 stimulated cell lysates. The benchmarking of mIP-tMS assays showed high correlation for quantitation of total target relative abundance compared to WB, ELISA and Luminex assays. However, for some phosphorylated targets, mIP-tMS assays had low concordance to the other immunoassays possibly due to differences in the specificity of anti-phospho antibodies used for each assay. Conclusion: Overall, the multiplex targeted MS assay can be used for identification and quantification of AKT/mTOR pathway proteins in cancer cell lines or tissue samples. Major advantages of this mIP-tMS assay are high confidence in target identity coupled with simultaneous quantitation of multiple targets and their PTMs. Citation Format: Bhavin Patel, Alex Behling, Leigh Foster, Ryan Bomgarden, Carrie Clothier, Kay Opperman, John Rogers. Quantitative analysis of IGF1R/AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3884.