Daniel Martin
M.D., Yale University School of Medicine


Phone: 206-732-1365
Office: 125

Email: dmartin@systemsbiology.org

Areas of Research:

The Martin laboratory´s research group has three distinct focuses: 1) identification of and test development for diagnostic markers for prostate cancer, 2) proteomic analysis of the androgen receptor to characterize the proteins participating in its signaling pathway, and 3) the development of innovative software tools for mass spectrometry-based proteomics.

Molecular Diagnostics.

Dr. Martin's research in the field of molecular diagnostics has focused on using tumor cell lines to generate candidate proteins for biomarkers. He has explored the "secretome" of prostate cancer cells using a mass spectrometry-based quantitative proteomic analysis. In his published work, he sought to identify prostate tumor cell proteins that are either "shed" or secreted into the extracellular environment and that may be of diagnostic value. In this context, he used a well-characterized prostate cancer cell line, LNCaP, and exploited the biochemical secretory program in these cells that is stimulated by androgenic hormones. The findings indicated that androgen-mediated release of proteins may occur through the activation of proteolytic enzymes rather than exclusively through transcriptional, translational, or secretory control mechanisms. Following up on the results described above, the laboratory has pursued a second generation of proteomic experiments to develop a more focused list of potential biomarker targets using a selective capture strategy, called glycocapture, that significantly improves the specificity of proteomic screening of cell lines. Glycocapture methods developed at the Institute for Systems Biology permit the covalent capture of proteins through N-linked carbohydrate side chains. These methods targets proteins that are destined for secretion and the extracellular membraneand that are glycosylated during post-translational processing in the endoplasmic reticulum and Golgi.

The Martin group has combined the glycocapture methodology with a cutting-edge mass spectrometry-based validation strategy called QCAT. This strategy utilizes a concatamer of the tryptic glycopeptides identified in the glycoprotein analysis for expression as an artificial epitope-tagged protein, which is purified and digested into peptides. This protein is expressed in isotopically heavy form, and thus each peptide is isotopically heavy. The collection of peptides can be spiked into serum for absolute quantification of the light peptides derived from an endogenous tumor. The analysis is performed using an ultra-sensitive mass spectrometry mode focused on only the peptides of interest. This method circumvents the problems of ELISA development, which can be expensive and time consuming. Its strengths include the ability to probe for cancer-derived proteins rather than identify global changes associated with disease, as is the case with top-down discovery methods. Success with this method could dramatically alter the field of biomarker discovery.

Most recently the laboratory has begun investigating the use of affinity capture reagents to enhance the sensitivity of mass spectrometry-based biomarker testing. We have initiated a program to select single chain antibody variable fragments (ScFv) from a yeast surface display library directed against peptides of interest. We will use these capture reagents to enrich for rare biomarker peptides in complex biospecimens and quantify them after release using mass spectrometry. Measurement will be assisted using isotopically heavy peptides produced using the QCAT strategy.

Proteomic Analyses of Transcription Factor Co-Regulators.

Dr. Martin has also focused on the characterization of the binding partners of the androgen receptor using proteomic methods. To date he has performed this analysis by immunoprecipitation of the wild-type androgen receptor and an epitope-tagged construct. Large-scale analyses are performed using stable isotope labeling of amino acids in cell culture (SILAC) using different washing and elution strategies to maintain the integrity of the protein complex. These experiments have identified a number of androgen receptor binding partners previously reported in the literature, including the chaperone proteins DnaJ, and HSP 70, as well as the coactivator proteins TRAP 150, non-POU domain containing proteins, octamer-binding proteins, and 14-3-3 protein zeta\delta. A number of unreported potential transcriptional activating proteins have also been identified which need molecular analysis for confirmation. The group is currently refining the purification techniques prior to proteomic analysis to increase the specificity of the list of targets that will be pursued. One such strategy includes crosslinking of proteins using formaldehyde to preserve protein complexes during purification.

Technology Development in Mass Spectrometry.

Dr. Martin´s interests in proteomics also include the study of some of the technical aspects of mass spectrometry. This work is overseen by Jimmy Eng, a senior computational biologist in Dr. Martin´s laboratory. Prior successful examples include 1) an analysis of the frequency and intensity of neutral loss during peptide fragmentation, 2) an analysis of the information content of immonium ion peaks, 3) development of a standard protein mix and database for instrument QC as well as software development, and 4) analysis of a unique digestion enzyme, Lys-N. More recently the lab has focused on the development of tools for targeted mass spectrometry using multiple reaction monitoring (MRM). This has resulted in the development of two software tools, MaRiMba and MRMer, as well as the publication of key observations and planning tools for MRM analysis.

The future directions of this research focus include the development of a new software tool, called ChromEval, for assessing chromatography quality, as well as a new tool for database searching using a graphics processing unit (video card) rather than a standard computer central processing unit (CPU).

Key collaborations within ISB:
Aimee Dudley
Jeff Ranish
Lee Hood

Key publications:

Zhang H, Li XJ, Martin DB, Aebersold R. Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 2003 21: 660-666.

Martin DB, Gifford DR, Wright ME, Keller A, Yi E, Goodlett DR, Aebersold R, Nelson PS. Quantitative proteomic analysis of proteins released by neoplastic prostate epithelium. Cancer Res. 2004 Jan 1;64(1):347-55.

Li XJ, Pedrioli PG, Eng J, Martin D, Yi EC, Lee H, Aebersold R. A tool to visualize and evaluate data obtained by liquid chromatography-electrospray ionization-mass spectrometry. Anal Chem. 2004 Jul 1;76(13):3856-60.

Eng JK, Martin DB, Aebersold R. (2004) Tandem mass spectrometry database searching. Encyclopedia of Genetics, Genomics, Proteomics and Bioinformatics, eds. Dunn M, Jorde L, Little P, and Subramaniam S, John Wiley & Sons, Ltd

Knudsen BS, Lucas JM, Fazli L, Hawley S, Falcon S, Coleman IM, Martin DB, Xu C, True LD, Gleave ME, Nelson PS, Ayala GE. Regulation of hepatocyte activator inhibitor-1 expression by androgen and oncogenic transformation in the prostate. Am J Pathol. 2005 Jul;167(1):255-66.

Martin DB, Eng JK, Nesvizhskii AI, Gemmill A, Aebersold R. Investigation of neutral loss during collision-induced dissociation of peptide ions. Anal Chem. 2005 Aug 1;77(15):4870-82.

Dall'Era MA, Oudes A, Martin DB, Liu AY. HSP27 and HSP70 interact with CD10 in C4-2 prostate cancer cells. Prostate. 2007 May 15;67(7):714-21

Klimek J, Eddes JS, Hohmann L, Jackson J, Peterson A, Letarte S, Gafken PR, Katz JE, Mallick P 3, Lee H, Schmidt A, Ossola R, Eng JK, Aebersold R, Martin DB. The Standard Protein Mix Database: A Diverse Data Set To Assist in the Production of Improved Peptide and Protein Identification Software Tools. J Proteome Res. 2007 Aug 2. PMID: 17711323

Schmid AK, Reiss DJ, Kaur A, Pan M, King N, Van PT, Hohmann L, Martin DB, Baliga NS. The anatomy of microbial cell state transitions in response to oxygen. Genome Res. 2007 Oct;17(10):1399-413. PMID: 17785531

Hohmann LJ, Eng JK, Gemmill A, Klimek J, Vitek O, Reid GE, Martin DB. Quantification of the Compositional Information Provided by Immonium Ions on a Quadrupole-Time-of-Flight Mass Spectrometer. Anal Chem 2008 Jul 15;80(14):5596-606. PMID: 18564857

Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, Lin DW, Urban N, Drescher CW, Knudsen BS, Stirewalt DL, Gentleman R, Vessella RL, Nelson PS, Martin DB, Tewari M. Circulating microRNAs as stable blood-based markers for cancer detection. M. Proc Natl Acad Sci U S A. 2008 Jul 29;105(30):10513-8. PMID: 18663219

Tabb DL, Ma ZQ, Martin DB, Ham AJ, Chambers MC. DirecTag: Accurate Sequence Tags from Peptide MS/MS through Statistical Scoring. J Proteome Res. 2008 Sep;7(9):3838-46.

Martin DB, Holzman T, May D, Peterson A, Eastham A, Eng J, McIntosh M. MRMer, an interactive open source and cross-platform system for data extraction and visualization of multiple reaction monitoring experiments. Mol Cell Proteomics. 2008 Nov;7(11):2270-8. PMID: 18641041.

Ventura CL, Higdon R, Hohmann L, Martin D, Kolker E, Liggitt HD, Skerrett SJ, Rubens CE. Staphylococcus aureus elicits marked alterations in the airway proteome during early pneumonia. Infect Immun. 2008 Dec;76(12):5862-72.

Hohmann L, Sherwood C, Eastham A, Peterson A, Eng JK, Eddes JS, Shteynberg D, Martin DB. Proteomic Analyses Using Grifola frondosa Metalloendoprotease Lys-N. J Proteome Res. 2009 Mar;8(3):1415-22. PMID: 19195997

Sherwood C, Eastham A, Lee LW, Risler J, Mirzaei H, Falkner JA, Martin DB. Rapid Optimization of MRM-MS Instrument Parameters by Subtle Alteration of Precursor and Product m/z Targets. J Proteome Res. 2009 Jul;8(7):3746-51. PMID: 19405522

Koide T, Reiss DJ, Bare JC, Pang WL, Facciotti MT, Schmid AK, Pan M, Marzolf B, Van PT, Lo FY, Pratap A, Deutsch EW, Peterson A, Martin D, Baliga NS. Prevalence of transcription promoters within archaeal operons and coding sequences. Mol Syst Biol. 2009;5:285. PMID: 19536208

Peterson A, Hohmann L, Huang L, Kim B, Eng JK, Martin DB. Analysis of RP-HPLC Loading Conditions for Maximizing Peptide Identifications in Shotgun Proteomics. 2009 Aug;8(8):4161-8. PMID: 19601632

Sherwood C, Eastham A, Peterson A, Eng JK, Shteynberg D, Mendoza L, Deutsch E, Risler J, Lee LW, Tasman N, Aebersold R, Lam H, Martin DB. MaRiMba: a Software Application for Spectral Library-Based MRM Transition List Assembly. J Proteome Res. 2009 Epub Jul 15. PMID: 1960382

Sherwood CA, Eastham A, Lee LW, Risler J, Vitek O, Martin DB. Correlation between y-Type Ions Observed in Ion Trap and Triple Quadrupole Mass Spectrometers. J Proteome Res. Epub 2009 Aug 12. PMID: 19603825

Why ISB?
Dr. Martin is interested in applying cutting-edge technology and systems-based methods to the clinical realm of oncology. He hopes to develop methods/insights that will lead to new diagnostic tools or identify possible targets for pharmacologic intervention.

	Group Personnel
			Executive Assistant Adrianna Wells
			Research Scientist Lee Adams Robert West Ted Whitmore
			Postdoctoral Fellow Ryan Austin Daniella Cohen
			Research Associate 2 Debbie Chang Jessica Weber Shile Zhang
			Research Associate 1 Ashleigh Baumgardner Carly Holstein Ian Sigmon