Oncogenic KRAS mutations are found in 40% of non-small cell lung carcinomas (NSCLC). In order to ... more Oncogenic KRAS mutations are found in 40% of non-small cell lung carcinomas (NSCLC). In order to expand the treatment options for NSCLC harboring oncogenic K-Ras, new therapeutic cell surface targets need to be identified and characterized. Towards this goal we carried out comparative cell surface analysis of the NSCLC cell line H2122-KRASG12C and the BL2122 cell line (i.e., control), which has been established from the peripheral blood lymphocytes of the same NSCLC patient. Here, we describe optimized hydrazide-based glycoproteomics for mapping of the cell surface proteome of the NSCLC H2122 cell line harboring oncogenic KRASG12C. Our comparative glycoproteomics revealed 632 proteins identified by LC-MS at the surface of both H2122-KRASG12C and BL2122 cell lines. Subtractive proteomics revealed 215 proteins detected solely at the H2122-KRASG12C cell surface while 214 proteins were found germane to the cell surface of BL2122 cells. A total of 203 proteins were commonly identified at the surface of both cell lines. Spectral counting based quantitation revealed 44 proteins showing ≥ 3-fold increase in their relative concentration at the cell surface of H2122-KRASG12C cells. Subsequent meta-analysis via Ingenuity Pathway Analysis (IPA) revealed significant activation of canonical pathways known to be involved in NSCLC biology (e.g., EGFR/neuregulin, and PI3K/AKT signaling). From a subset of proteins showing significant up-regulation at the surface of H2122-KRASG12C cells, we further cross-validated CD147 using immunofluorescence analysis (IFA) and Western blotting (WB). Interestingly, subsequent IFA confirmed the over-expression of CD147 at the cell surface of pancreatic KP-3, lung H2444, and colon SW620 cancer cell lines, each harboring constitutively activated KRAS. Importantly, amongst 215 proteins identified solely at the cell surface of H2122-KRASG12C cells, proteins upstream of K-Ras, epidermal growth factor receptor (EGFR), receptor tyrosine-protein kinase erbB-2 (ERBB2), receptor tyrosine-protein kinase erbB-3 (ERBB3), and disintegrin metalloproteinase domain-containing protein 17 (ADAM17) were unambiguously identified. Using WB analysis, we first confirmed the expression K-Ras in the membrane preparation of H2122-KRASG12C cells. Interestingly, insulin-like growth factor 1 receptor, (IGF1R), and mesothelin (MSLN) were also detected exclusively at the cell surface of the H2122-KRASG12C. We further cross-validated the expression of mesothelin using WB. Taken together, present approach greatly extends the known cell surface phenotype of the NSCLC H2122-KRASG12C cells and can be readily employed as a primary proteomic screen to provide the basis for discovery and characterization of novel cell surface therapeutic targets or diagnostic assays in cells/tissues harboring oncogenic K-Ras. Citation Format: Xiaoying Ye, Thomas J. Turbyville, Rachel Bagni, Franck McCormick, Gordon Whiteley, Josip Blonder. Comparative surface proteomics of NCI-H2122 cells reveals distinct cell surface phenotype of a metastatic NSCLC cell line expressing oncogenic KRASG12C. [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 1829. doi:10.1158/1538-7445.AM2015-1829
Biochimica Et Biophysica Acta - Proteins And Proteomics, Apr 1, 2004
We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-... more We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-charge (m/z) dimension (GPF(m/z)) for increasing the effective number of protein identifications in cases where sample quantity limits the use of multi-dimensional chromatographic fractionation. A peptide digestate from proteins isolated from the membrane fraction of natural killer (NK) cells was analyzed by microcapillary reversed-phase liquid chromatography coupled online to an ion-trap (IT) mass spectrometer. Performing GPF(m/z) using eight narrow precursor ion scan m/z ranges enabled the identification of 340 NK cell proteins from 12 microg of digestate, representing more than a fivefold increase in the number of proteins identified as compared to the same experiment employing a standard precursor ion survey scan m/z range (i.e., m/z 400-2000). The results show that GPF(m/z) represents an effective technique for increasing protein identifications in global proteomic investigations especially when sample quantity is limited.
The search for disease markers is not new; however, with the emergence of new technologies such a... more The search for disease markers is not new; however, with the emergence of new technologies such as nano-HPLC and electrospray ionization and time of flight mass spectrometry, the search has intensified considerably. Genomic, proteomic and metabolomic technologies are being used to search for novel disease markers. In this manuscript emphasis will be on different HPLC and MS methods that are used to search for metabolites and proteins that can be used for the discovery of novel, sensitive and specific disease biomarkers. Definitions of terms such as sensitivity, specificity, and protein profiles will be given. Methods used for effective fractionation, separation and quantitation of proteins and peptides using HPLC/MS will be discussed and examples are presented. A brief discussion of electrophoretic procedures used for protein fractionation and biomarker discovery is also included.
A major goal of proteomics is to develop methods that enable the systematic characterization of e... more A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.
Evaluation of: Mallick P, Schirle M, Chen SS et al. Computational prediction of proteotypic pepti... more Evaluation of: Mallick P, Schirle M, Chen SS et al. Computational prediction of proteotypic peptides for quantitative proteomics. Nat. Biotechnol. 25(1), 125–131 (2007). Mass spectrometry, the driving analytical force behind proteomics, is primarily used to identify and quantify as many proteins in a complex biological mixture as possible. While there are many ways to prepare samples, one aspect that is common to a vast majority of bottom-up proteomic studies is the digestion of proteins into tryptic peptides prior to their analysis by mass spectrometry. As correctly highlighted by Mallick and colleagues, only a few peptides are repeatedly and consistently identified for any given protein within a complex mixture. While the existence of these proteotypic peptides (to borrow the authors’ terminology) is well known in the proteomics community, there has never been an empirical method to recognize which peptides may be proteotypic for a given protein. In this study, the investigators discovered over 16,000 proteotypic peptides from a collection of over 600,000 peptide identifications obtained from four different analytical platforms. The study examined a number of physicochemical parameters of these peptides to determine which properties were most relevant in defining a proteotypic peptide. These characteristic properties were then used to develop computational tools to predict proteotypic peptides for any given protein within an organism.
The previous decade witnessed an enormous number of studies with the singular goal of identifying... more The previous decade witnessed an enormous number of studies with the singular goal of identifying protein biomarkers for diseases such as cancer. A large majority of these studies have focused on comparative studies of serum or plasma obtained from disease‐affected and control patients. In these studies, proteins identified in the samples using MS were compared with the hope that differences between samples would reveal useful biomarkers. Unfortunately, finding clinically relevant biomarkers has often been elusive and frustrating. As with most research efforts, both successes and failures, much has been learned about what strategies work and which do not. Part of the problem can be attributed to underestimating the effort required to discover novel biomarkers and depending too heavily on MS analysis of peripheral blood samples. Fortunately, the future for biomarker discovery still appears bright. MS technology continues to increase in sensitivity, throughput, and accuracy while novel types of samples and clever experimental designs coupled with innovative bioinformatics will make this vision of routine biomarker discovery a reality. To achieve ultimate success is going to require concomitant application of a number of different technologies, all providing the information necessary for discovering and validating clinically useful biomarkers.
This study describes the application of a single tube sample preparation technique coupled with m... more This study describes the application of a single tube sample preparation technique coupled with multidimensional fractionation for the analysis of a complex membrane protein sample from murine natural killer (NK) cells. A solution-based method that facilitates the solubilization and tryptic digestion of integral membrane proteins is conjoined with strong cation exchange (SCX) liquid chromatography (LC) fractionation followed by microcapillary reversed-phase (microRP) LC tandem mass spectrometric analysis of each SCXLC fraction in second dimension. Sonication in buffered methanol solution was employed to solubilize, and tryptically digest murine NK cell microsomal proteins, allowing for the large-scale identification of integral membrane proteins, including the mapping of the membrane-spanning peptides. Bioinformatic analysis of the acquired tandem mass spectra versus the murine genome database resulted in 11,967 matching tryptic peptide sequences, corresponding to 5782 unique peptide identifications. These peptides resulted in identification of 2563 proteins of which 876 (34%) are classified as membrane proteins.
Although proteomic technology has proved to be extremely powerful in basic research, its impact h... more Although proteomic technology has proved to be extremely powerful in basic research, its impact has not been as great in the clinical laboratory. The future, however, looks extremely positive because technologies, such as mass spectrometry and tissue microarrays, have continued to improve over the past several years. One of the most exciting developments, particularly in the area of mass spectrometry, is the ability to examine formalin-fixed paraffin-embedded tissue using these technologies. The almost inexhaustible supply of these tissues will enable proteomic laboratories access to clinically important specimens that will undoubtedly lead to a number of important discoveries in the near future.
The extracellular matrix (ECM) is the connective tissue component generated and secreted by cells... more The extracellular matrix (ECM) is the connective tissue component generated and secreted by cells to provide structural and functional support, while extracellular vesicles are distinct membrane-enclosed structures present outside of eucaryotic cells that carry out distinct biological functions. Different cell types release distinct populations of vesicles that fulfill various functions. Exosomes are vesicles commonly secreted by a variety of cells, whereas matrix vesicles (MVs) are specifically produced and secreted by bone cells to facilitate the formation of the ECM. This article focuses on the characteristics of the ECM and extracellular vesicles, and reviews the latest progress in applying proteomic technologies to analyze these features. The findings and implications in developmental biology, tumor biology, immunology, biomarker discovery, and vaccine research are also discussed.
A combined, detergent‐ and organic solvent‐based proteomic method for the analysis of detergent‐r... more A combined, detergent‐ and organic solvent‐based proteomic method for the analysis of detergent‐resistant membrane rafts (DRMR) is described. These specialized domains of the plasma membrane contain a distinctive and dynamic protein and/or lipid complement, which can be isolated from most mammalian cells. Lipid rafts are predominantly involved in signal transduction and adapted to mediate and produce different cellular responses. To facilitate a better understanding of their biology and role, DRMR were isolated from Vero cells as a Triton X‐100 insoluble fraction. After detergent removal, sonication in 60% buffered methanol was used to extract, solubilize and tryptically digest the resulting protein complement. The peptide digestate was analyzed by microcapillary reversed‐phase liquid chromatography‐tandem mass spectrometry. Gas‐phase fractionation in the mass‐to‐charge range was employed to broaden the selection of precursor ions and increase the number of identifications in an effort to detect less abundant proteins. A total of 380 proteins were identified including all known lipid raft markers. A total of 91 (24%) proteins were classified as integral α‐helical membrane proteins, of which 51 (56%) were predicted to have multiple transmembrane domains.
Oncogenic KRAS mutations are found in 40% of non-small cell lung carcinomas (NSCLC). In order to ... more Oncogenic KRAS mutations are found in 40% of non-small cell lung carcinomas (NSCLC). In order to expand the treatment options for NSCLC harboring oncogenic K-Ras, new therapeutic cell surface targets need to be identified and characterized. Towards this goal we carried out comparative cell surface analysis of the NSCLC cell line H2122-KRASG12C and the BL2122 cell line (i.e., control), which has been established from the peripheral blood lymphocytes of the same NSCLC patient. Here, we describe optimized hydrazide-based glycoproteomics for mapping of the cell surface proteome of the NSCLC H2122 cell line harboring oncogenic KRASG12C. Our comparative glycoproteomics revealed 632 proteins identified by LC-MS at the surface of both H2122-KRASG12C and BL2122 cell lines. Subtractive proteomics revealed 215 proteins detected solely at the H2122-KRASG12C cell surface while 214 proteins were found germane to the cell surface of BL2122 cells. A total of 203 proteins were commonly identified at the surface of both cell lines. Spectral counting based quantitation revealed 44 proteins showing ≥ 3-fold increase in their relative concentration at the cell surface of H2122-KRASG12C cells. Subsequent meta-analysis via Ingenuity Pathway Analysis (IPA) revealed significant activation of canonical pathways known to be involved in NSCLC biology (e.g., EGFR/neuregulin, and PI3K/AKT signaling). From a subset of proteins showing significant up-regulation at the surface of H2122-KRASG12C cells, we further cross-validated CD147 using immunofluorescence analysis (IFA) and Western blotting (WB). Interestingly, subsequent IFA confirmed the over-expression of CD147 at the cell surface of pancreatic KP-3, lung H2444, and colon SW620 cancer cell lines, each harboring constitutively activated KRAS. Importantly, amongst 215 proteins identified solely at the cell surface of H2122-KRASG12C cells, proteins upstream of K-Ras, epidermal growth factor receptor (EGFR), receptor tyrosine-protein kinase erbB-2 (ERBB2), receptor tyrosine-protein kinase erbB-3 (ERBB3), and disintegrin metalloproteinase domain-containing protein 17 (ADAM17) were unambiguously identified. Using WB analysis, we first confirmed the expression K-Ras in the membrane preparation of H2122-KRASG12C cells. Interestingly, insulin-like growth factor 1 receptor, (IGF1R), and mesothelin (MSLN) were also detected exclusively at the cell surface of the H2122-KRASG12C. We further cross-validated the expression of mesothelin using WB. Taken together, present approach greatly extends the known cell surface phenotype of the NSCLC H2122-KRASG12C cells and can be readily employed as a primary proteomic screen to provide the basis for discovery and characterization of novel cell surface therapeutic targets or diagnostic assays in cells/tissues harboring oncogenic K-Ras. Citation Format: Xiaoying Ye, Thomas J. Turbyville, Rachel Bagni, Franck McCormick, Gordon Whiteley, Josip Blonder. Comparative surface proteomics of NCI-H2122 cells reveals distinct cell surface phenotype of a metastatic NSCLC cell line expressing oncogenic KRASG12C. [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 1829. doi:10.1158/1538-7445.AM2015-1829
Biochimica Et Biophysica Acta - Proteins And Proteomics, Apr 1, 2004
We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-... more We have explored the utility of gas-phase fractionation by mass spectrometry (MS) in the mass-to-charge (m/z) dimension (GPF(m/z)) for increasing the effective number of protein identifications in cases where sample quantity limits the use of multi-dimensional chromatographic fractionation. A peptide digestate from proteins isolated from the membrane fraction of natural killer (NK) cells was analyzed by microcapillary reversed-phase liquid chromatography coupled online to an ion-trap (IT) mass spectrometer. Performing GPF(m/z) using eight narrow precursor ion scan m/z ranges enabled the identification of 340 NK cell proteins from 12 microg of digestate, representing more than a fivefold increase in the number of proteins identified as compared to the same experiment employing a standard precursor ion survey scan m/z range (i.e., m/z 400-2000). The results show that GPF(m/z) represents an effective technique for increasing protein identifications in global proteomic investigations especially when sample quantity is limited.
The search for disease markers is not new; however, with the emergence of new technologies such a... more The search for disease markers is not new; however, with the emergence of new technologies such as nano-HPLC and electrospray ionization and time of flight mass spectrometry, the search has intensified considerably. Genomic, proteomic and metabolomic technologies are being used to search for novel disease markers. In this manuscript emphasis will be on different HPLC and MS methods that are used to search for metabolites and proteins that can be used for the discovery of novel, sensitive and specific disease biomarkers. Definitions of terms such as sensitivity, specificity, and protein profiles will be given. Methods used for effective fractionation, separation and quantitation of proteins and peptides using HPLC/MS will be discussed and examples are presented. A brief discussion of electrophoretic procedures used for protein fractionation and biomarker discovery is also included.
A major goal of proteomics is to develop methods that enable the systematic characterization of e... more A major goal of proteomics is to develop methods that enable the systematic characterization of every protein within the cell or particular subcellular proteome using a single analytical platform. Although the equivalent has already been achieved in genomics, reaching this goal in proteomics represents a much greater challenge due to the wide dynamic range of protein expression, numerous post-translational modifications and remarkable physicochemical heterogeneity of proteins. A major analytical challenge has involved developing more effective means for proteome-scale investigations of membrane proteins, whose solubility differs drastically from that of cytoplasmic proteins. Fortunately, rapid progress has increased the ability to characterize this critically important class of proteins on a scale analogous to that of aqueous soluble proteins.
Evaluation of: Mallick P, Schirle M, Chen SS et al. Computational prediction of proteotypic pepti... more Evaluation of: Mallick P, Schirle M, Chen SS et al. Computational prediction of proteotypic peptides for quantitative proteomics. Nat. Biotechnol. 25(1), 125–131 (2007). Mass spectrometry, the driving analytical force behind proteomics, is primarily used to identify and quantify as many proteins in a complex biological mixture as possible. While there are many ways to prepare samples, one aspect that is common to a vast majority of bottom-up proteomic studies is the digestion of proteins into tryptic peptides prior to their analysis by mass spectrometry. As correctly highlighted by Mallick and colleagues, only a few peptides are repeatedly and consistently identified for any given protein within a complex mixture. While the existence of these proteotypic peptides (to borrow the authors’ terminology) is well known in the proteomics community, there has never been an empirical method to recognize which peptides may be proteotypic for a given protein. In this study, the investigators discovered over 16,000 proteotypic peptides from a collection of over 600,000 peptide identifications obtained from four different analytical platforms. The study examined a number of physicochemical parameters of these peptides to determine which properties were most relevant in defining a proteotypic peptide. These characteristic properties were then used to develop computational tools to predict proteotypic peptides for any given protein within an organism.
The previous decade witnessed an enormous number of studies with the singular goal of identifying... more The previous decade witnessed an enormous number of studies with the singular goal of identifying protein biomarkers for diseases such as cancer. A large majority of these studies have focused on comparative studies of serum or plasma obtained from disease‐affected and control patients. In these studies, proteins identified in the samples using MS were compared with the hope that differences between samples would reveal useful biomarkers. Unfortunately, finding clinically relevant biomarkers has often been elusive and frustrating. As with most research efforts, both successes and failures, much has been learned about what strategies work and which do not. Part of the problem can be attributed to underestimating the effort required to discover novel biomarkers and depending too heavily on MS analysis of peripheral blood samples. Fortunately, the future for biomarker discovery still appears bright. MS technology continues to increase in sensitivity, throughput, and accuracy while novel types of samples and clever experimental designs coupled with innovative bioinformatics will make this vision of routine biomarker discovery a reality. To achieve ultimate success is going to require concomitant application of a number of different technologies, all providing the information necessary for discovering and validating clinically useful biomarkers.
This study describes the application of a single tube sample preparation technique coupled with m... more This study describes the application of a single tube sample preparation technique coupled with multidimensional fractionation for the analysis of a complex membrane protein sample from murine natural killer (NK) cells. A solution-based method that facilitates the solubilization and tryptic digestion of integral membrane proteins is conjoined with strong cation exchange (SCX) liquid chromatography (LC) fractionation followed by microcapillary reversed-phase (microRP) LC tandem mass spectrometric analysis of each SCXLC fraction in second dimension. Sonication in buffered methanol solution was employed to solubilize, and tryptically digest murine NK cell microsomal proteins, allowing for the large-scale identification of integral membrane proteins, including the mapping of the membrane-spanning peptides. Bioinformatic analysis of the acquired tandem mass spectra versus the murine genome database resulted in 11,967 matching tryptic peptide sequences, corresponding to 5782 unique peptide identifications. These peptides resulted in identification of 2563 proteins of which 876 (34%) are classified as membrane proteins.
Although proteomic technology has proved to be extremely powerful in basic research, its impact h... more Although proteomic technology has proved to be extremely powerful in basic research, its impact has not been as great in the clinical laboratory. The future, however, looks extremely positive because technologies, such as mass spectrometry and tissue microarrays, have continued to improve over the past several years. One of the most exciting developments, particularly in the area of mass spectrometry, is the ability to examine formalin-fixed paraffin-embedded tissue using these technologies. The almost inexhaustible supply of these tissues will enable proteomic laboratories access to clinically important specimens that will undoubtedly lead to a number of important discoveries in the near future.
The extracellular matrix (ECM) is the connective tissue component generated and secreted by cells... more The extracellular matrix (ECM) is the connective tissue component generated and secreted by cells to provide structural and functional support, while extracellular vesicles are distinct membrane-enclosed structures present outside of eucaryotic cells that carry out distinct biological functions. Different cell types release distinct populations of vesicles that fulfill various functions. Exosomes are vesicles commonly secreted by a variety of cells, whereas matrix vesicles (MVs) are specifically produced and secreted by bone cells to facilitate the formation of the ECM. This article focuses on the characteristics of the ECM and extracellular vesicles, and reviews the latest progress in applying proteomic technologies to analyze these features. The findings and implications in developmental biology, tumor biology, immunology, biomarker discovery, and vaccine research are also discussed.
A combined, detergent‐ and organic solvent‐based proteomic method for the analysis of detergent‐r... more A combined, detergent‐ and organic solvent‐based proteomic method for the analysis of detergent‐resistant membrane rafts (DRMR) is described. These specialized domains of the plasma membrane contain a distinctive and dynamic protein and/or lipid complement, which can be isolated from most mammalian cells. Lipid rafts are predominantly involved in signal transduction and adapted to mediate and produce different cellular responses. To facilitate a better understanding of their biology and role, DRMR were isolated from Vero cells as a Triton X‐100 insoluble fraction. After detergent removal, sonication in 60% buffered methanol was used to extract, solubilize and tryptically digest the resulting protein complement. The peptide digestate was analyzed by microcapillary reversed‐phase liquid chromatography‐tandem mass spectrometry. Gas‐phase fractionation in the mass‐to‐charge range was employed to broaden the selection of precursor ions and increase the number of identifications in an effort to detect less abundant proteins. A total of 380 proteins were identified including all known lipid raft markers. A total of 91 (24%) proteins were classified as integral α‐helical membrane proteins, of which 51 (56%) were predicted to have multiple transmembrane domains.
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Papers by Josip Blonder