TECHNICAL BRIEF NanoLC-FT-ICR MS improves proteome coverage attainable for ,3000 laser-microdissected breast carcinoma cells Arzu Umar 1 , Theo M. Luider 2 , John A. Foekens 1 and Ljiljana Pas ˇa-Tolic ´ 3 1 Department of Medical Oncology, Erasmus Medical Center Rotterdam, Josephine Nefkens Institute, The Netherlands 2 Department of Neurology, Erasmus Medical Center Rotterdam, Josephine Nefkens Institute, The Netherlands 3 Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA Proteomics assays hold great promise for unraveling molecular events that underlie human dis- eases. Effective analysis of clinical samples is essential, but this task is considerably complicated by tissue heterogeneity. Laser capture microdissection (LCM) can be used to selectively isolate target cells from their native tissue environment. However, the small number of cells that is typically procured by LCM severely limits proteome coverage and biomarker discovery potential achievable by conventional proteomics platforms. Herein, we describe the use of nanoLC-FT-ICR MS for analyzing protein digests of ,3000 LCM-derived tumor cells from breast carcinoma tissue, corresponding to ,300 ng of total protein. A total of 2282 peptides were identified by matching LC-MS data to accurate mass and time (AMT) tag databases that were previously established for human breast (cancer) cell lines. One thousand and three unique proteins were confidently identified with two or more peptides. Based on gene ontology categorization, identi- fied proteins appear to cover a wide variety of biological functions and cellular compartments. This work demonstrates that a substantial number of proteins can be detected and identified from limited number of cells using the AMT tag approach, and opens doors for high-throughput in-depth proteomics analysis of clinical samples. Received: April 19, 2006 Revised: October 10, 2006 Accepted: October 14, 2006 Keywords: Accurate mass and time tag / Breast cancer / Fourier transform-ion cyclotron reso- nance mass spectrometry / Laser capture microdissection / Nanoscale liquid chroma- tography Proteomics 2007, 7, 323–329 323 Breast cancer is the most common malignancy in women in the Western World. While this cancer can be effectively treated at the primary stage in a majority of cases [1, 2], patients with recurrent breast cancer have a very low 5-year survival rate due to cancer resistance to therapy [3, 4]. Since the introduction of gene expression microarrays, an enormous effort has been put forth to identify improved biomarkers or marker profiles that aid in early diagnosis, therapy prediction, and patient prognosis [5–7]. As powerful as gene expression data have proven to be, protein-level information may actually be the missing link needed to translate molecular knowledge into clinical applications. Correspondence: Dr. Arzu Umar, Erasmus Medical Center Rotter- dam, Josephine Nefkens Institute, Department of Medical Oncol- ogy, Dr. Molewaterplein 50, Be 428 PO BOX 2040, 3000 CA, Rot- terdam, The Netherlands E-mail: a.umar@erasmusmc.nl Fax: +31-10-4088365 Abbreviations: AMT , accurate mass and time; FDR, false discov- ery rate; HMEC, human mammary epithelial cell line; LCM, laser capture microdissection; LMPC, laser microdissection and pres- sure catapulting; MMA, mass measurement accuracy; NET, nor- malized (chromatographic) elution time; TMA, time measure- ment accuracy; UMC, unique mass classes DOI 10.1002/pmic.200600293  2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com