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


Phone: 206-732-1365
Office: 125

Email: dmartin@systemsbiology.org

Areas of Research:

Dr. Martin's laboratory research to date has focused on application of proteomics in three distinct spheres. The first is to use cutting edge instrumentation to identify new diagnostic markers for prostate cancer. The second is to perform a focused proteomic analysis on the androgen receptor to characterize the proteins participating in this signaling pathway. Finally Dr. Martin has been fascinated by mass spectrometry instrumentation, mechanisms of peptide fragmentation, and computational aspects of mass spectrometry-based proteomics and have pursued a variety of small projects along these lines.

A) 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. To quantify secreted/shed proteins, isotopically light and heavy ICAT reagents were used. Proteomic analysis of the LNCaP media identified in excess of 600 proteins, 524 of which could be quantified. Ten percent of the proteins identified were androgen regulated and a subset of these proteins appeared to be expressed in abundance. Selected mass spectrometry observations were confirmed by Western analysis. 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. In collaboration with Dr. Knudsen at FHCRC, we demonstrated that hepatocyte growth factor activator inhibitor 1 (HAI-1) is over expressed in primary prostate carcinoma.

Following up on the results described above, Dr. Martin has begun a second generation of proteomic experiments to develop a more focused list of potential biomarker targets using a selective capture strategy that significantly improves the specificity of proteomic screening of cell lines. Methods developed at the Institute for Systems Biology permit the covalent capture of proteins through N-linked carbohydrate side chains. These methods targets proteins destined for secretion and the extracellular membrane, which are glycosylated during post translational processing in the endoplasmic reticulum and Golgi. Two such glycocapture methodologies are currently being applied to prostate cancer cell lines. In the first, extracellular membrane proteins are biotinylated on live cells through the carbohydrate groups for capture by streptavidin. The second method captures N-linked glycoproteins in solution from culture media using an immobilized hydrazide chemistry. In both of these methods, proteins are digested to peptides which are released from the resin by cleaving the bond between sugar and the asparagine residue. Liberated peptides are analyzed using mass spectrometry. 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, thus each peptide is isotopically heavy. The collection of peptides can be spiked into human 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.

With this list in hand, we will employ 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 bacteria grown in isotopically heavy ammonium chloride supplemented minimal media, and thus each peptide is isotopically heavy. The collection of peptides can be spiked into human 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.

B) 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 had performed this analysis by immunoprecipitation of the wild type and an epitope tagged construct. Thus far, three successful large scale analyses have been performed using stable isotope tagging 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 binding partners previously reported in the literature, including the chaperone proteins DnaJ homolog C, HSP 70, as well as the coactivator proteins TRAP 150, non-POU domain containing, octamer-binding proteins, and 14-3-3 protein zeta\delta. A number of unreported potential transcriptional activating proteins have been identified which need molecular analysis for confirmation. He is currently refining the purification techniques prior to proteomic analysis to increase the specificity of the list of targets which will be pursued. One such strategy includes crosslinking of proteins using formaldehyde to preserve protein complexes during purification. Preliminary work using an epitope tagged androgen receptor shows promise.

C) Technology Development in Mass Spectrometry.
Software/Analysis: Dr. Martin´s interests in proteomics also include study of some of the technical aspects of mass spectrometry. Recently while examining mass spectra from a large experiment, he noticed the frequent appearance of unmatched fragment peaks. Working with Jimmy Eng of the ISB and FHCRC, Dr. Martin was able to perform an analysis of the frequency and intensity of neutral loss during peptide fragmentation, which to his pleasant surprise produced a publication. This will soon be followed up with papers currently in preparation describing 1) the presence of another type of fragment ion, the immonium ion, in TOF spectra and 2) the spectral features associated with digestion using a unique proteolytic enzyme. Finally, Dr. Martin has been involved in the development of a set of small molecule standards that can be added to the HPLC solvents which improve the tracking of retention time runs during introduction of sample into the mass spectrometer. This work appears to be quite promising and software is under development to determine the effectiveness of this approach.

Key collaborations within ISB:
Jeff Ranish
Lee Hood

Key publications:

Piening B, Whiteaker J, Zhang H, Schaffer SA, Martin D, Hohmann L, Cooke K, Olson J, Hansen S, Flory MR, Lee H, Watts J, Goodlett DR, Aebersold R, Paulovich A, Schwikowski B, Prakash A.
Assessing bias in experiment design for large-scale mass spectrometry-based quantitative proteomics. Mol Cell Proteomics. 2007 Jul 7

Dall'era MA, Oudes A, Martin DB, Liu AY.
HSP27 and HSP70 interact with CD10 in C4-2 prostate cancer cells Prostate. 2007 Mar 6;

Mallick P, Schirle M, Chen SS, Flory MR, Lee H, Martin D, Ranish J, Raught B, Schmitt R, Werner T, Kuster B, Aebersold R.
Computational prediction of proteotypic peptides for quantitative proteomics Nat Biotechnol. 2006 Dec 31

King NL, Deutsch EW, Ranish JA, Nesvizhskii AI, Eddes JS, Mallick P, Eng J, Desiere F, Flory M, Martin DB, Kim B, Lee H, Raught B, Aebersold R.
Genome Biol. 2006 Nov 13;7(11):R106

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.

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.

Desiere F, Deutsch EW, Nesvizhskii AI, Mallick P, King NL, Eng JK, Aderem A, Boyle R, Brunner E, Donohoe S, Fausto N, Hafen E, Hood L, Katze MG, Kennedy KA, Kregenow F, Lee H, Lin B, Martin D, Ranish JA, Rawlings DJ, Samelson LE, Shiio Y, Watts JD, Wollscheid B, Wright ME, Yan W, Yang L, Yi EC, Zhang H, Aebersold R.
Integration with the human genome of peptide sequences obtained by high-throughput mass spectrometry. Genome Biol. 2005;6(1):R9.

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.

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.

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.

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 Lik Wee Lee
			Research Associate 2 Debbie Chang Jessica Weber Shile Zhang
			Research Associate 1 Ashleigh Baumgardner Carly Holstein Ian Sigmon