Faculty Bibliography

In this article, we present an overview of the different strategies for sample preparation for identification by mass spectrometry (MS) of biomarkers from serum and/or plasma. We consider the effects of the variables involved in sample collection, handling and storage, and describe different approaches for removal of high abundance proteins and serum/plasma fractionation. We review the advantages and disadvantages of such techniques as centrifugal ultrafiltration, different formats for solid phase extraction, organic solvent extraction, gel and capillary electrophoresis, and liquid chromatography. We also discuss a variety of current proteomic methods and their main applications for biomarker-related studies

Cysteine sulfenic acid (Cys-SOH) is an elusive intermediate in reactive oxygen species-induced oxidation reactions of many proteins such as peroxiredoxins and tyrosine phosphatases. Cys-SOH is proposed to play a vital role in catalytic and signaling functions. The formation of cysteine sulfinic acid (Cys-SO(2)H) and cysteine sulfonic acid (Cys-SO(3)H) has been implicated in the activation of matrix metalloproteinase-7 (MMP-7) and oxidation of thiol to cysteine sulfinic acid has been associated with the autolytic cleavage of MMP-7. We have examined the formation of cysteine sulfenic acid in a synthetic peptide PRCGVPDVA, which is a cysteine switch domain of MMP-7 and other matrix metalloproteases. We have prepared the cysteine sulfenic acid containing peptide, PRC(SOH)GVPDVA, by reaction with hydroxyl radicals generated by the Fenton reaction (Fe(+2)/H(2)O(2)). We characterized this modified peptide by tandem mass spectrometry and accurate mass measurement experiments. In addition, we used 7-chloro-4-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl) reagent to form an adduct with PRC(SOH)GVPDVA to provide additional evidence for the viability of PRC(SOH)GVPDVA in solution. We also characterized an intramolecular cysteine sulfinamide cross-link product PRC[S(O)N]GVPDVA based on tandem mass spectrometry and accurate mass measurement experiments. These results contribute to the understanding of a proteolytic cleavage mechanism that is traditionally associated with MMP activation

Both the generation and the analysis of proteomics data are now widespread, and high-throughput approaches are commonplace. Protocols continue to increase in complexity as methods and technologies evolve and diversify. To encourage the standardized collection, integration, storage and dissemination of proteomics data, the Human Proteome Organization's Proteomics Standards Initiative develops guidance modules for reporting the use of techniques such as gel electrophoresis and mass spectrometry. This paper describes the processes and principles underpinning the development of these modules; discusses the ramifications for various interest groups such as experimentalists, funders, publishers and the private sector; addresses the issue of overlap with other reporting guidelines; and highlights the criticality of appropriate tools and resources in enabling 'MIAPE-compliant' reporting

Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly

Identification of tyrosine phosphorylation by MS is challenging due to its low abundance in biological samples. Therefore, specific enrichment of tyrosine phosphorylated peptides prior to their analysis is highly desirable. The application of immunopurification of phosphotyrosine (pY) peptides using pY antibodies has been greatly limited by poor selectivity. In the present study, we have shown that the selectivity of pY peptide immunopurification can be dramatically improved by adding detergents to immunoprecipitation buffers. Optimum selectivity and sensitivity were achieved using an immunoprecipitation buffer containing n-octyl glucoside with a concentration above its critical micelle concentration (0.7%). The optimized method was used to identify in vivo tyrosine phosphorylation on proteins isolated from cell extract by anti-pY protein immunoprecipitation. After immunopurification, non-pY-containing peptides from protein digests were readily removed and pY peptides became the dominant peaks in MALDI quadrupole-TOF mass spectra. In addition, the signal intensities from pY-containing peptides were enhanced significantly after enrichment, allowing characterization of tyrosine phosphorylation sites with greater sensitivity

Comparative proteomic approaches using isotopic labeling and mass spectrometry (MS) have become increasingly popular. Conventionally, quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with 13C6 lysine and 15N4 arginine, the other set unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a ten-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG-108 cells

We present an improved method for MALDI-MS analysis of proteins that have been electroblotted onto a nitrocellulose (NC) membrane. With this approach, electroblotted proteins can be analyzed directly for intact molecular weight determination or after on-membrane digestion by dissolution of the nitrocellulose in MALDI matrix solution containing 70% acetonitrile and 30% methanol. This solution helps maintain solubility of proteins and peptides while dissolving the NC membrane, which is dissolved by 100% acetone in other protocols. On-membrane tryptic digestion using this method requires half the time of in-gel digestion and results in fewer missed cleavages and better protein coverage. For the membrane proteins studied, bovine uroplakins II and III, the protein coverage was almost twice that provided by conventional in-gel digestion, and the transmembrane domains of both uroplakins were detected only after on-membrane digestion. We also demonstrated the compatibility with MALDI-MS of a new dye, MemCode, which is specifically designed for staining NC membrane-immobilized proteins and is faster and more sensitive than Ponceau-S. Our improved on-membrane digestion protocol greatly improves the study of soluble and, particularly strikingly, integral membrane proteins by mass spectrometry

Eph-related receptor tyrosine kinases (RTK) have been implicated in several biological functions including synaptic plasticity, axon guidance, and morphogenesis, yet the details of the signal transduction pathways that produce these specific biological functions after ligand-receptor interaction remain unclear. We used Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) in combination with LC-MS/MS to characterize cellular signaling following stimulation by ephrinB1-Fc of NG-108 cells that overexpress EphB2 receptors. Because tyrosine phosphorylation functions as a key regulatory event in RTK signaling, we used anti-phosphotyrosine immunoprecipitation (pY IP) of cell lysates to isolate potential participants in the EphB2 pathway. Our SILAC experiments identified 127 unique proteins, 40 of which demonstrated increased abundance in pY IPs from ephrinB1-Fc stimulated cells as compared with unstimulated cells. Six proteins demonstrated decreased abundance, and 81 did not change significantly in relative abundance. Western blotting analysis of five proteins after pY IP verified their SILAC results. On the basis of previously published work and use of PathwayAssist software, we proposed an interaction network downstream of EphB2 for the proteins with changed ratios