412 Proteomics Clin. Appl. 2012, 6, 412–417 DOI 10.1002/prca.201200016 TECHNICAL BRIEF Proteomic approach to human kidney glomerulus prepared by laser microdissection from frozen biopsy specimens: Exploration of proteome after removal of blood-derived proteins Yutaka Yoshida 1 , Masaaki Nameta 1 , Masayoshi Kuwano 2 , Ying Zhang 1 , Xu Bo 1 , Sameh Magdeldin 1,3 , Zenyui Cui 1 , Hidehiko Fujinaka 1,4 , Eishin Yaoita 1 , Takeshi Tomonaga 2 and Tadashi Yamamoto 1 1 Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, Japan 2 Laboratory of Proteome Research, National Institute of Biomedical Innovation, Japan 3 Department of Physiology, Faculty of Veterinary Medicine, Suez Canal University, Egypt 4 Institute of Clinical Research, Niigata National Hospital, Japan Purpose: Abundance of blood-derived proteins in glomeruli prepared by laser microdissection from human kidney biopsy specimens has hampered in-depth proteomic analysis of glomeruli. We attempted to establish experimental platform for in-depth proteomic analysis of glomeruli by removal of blood-derived proteins from frozen biopsy samples. Experimental design: Frozen sections of biopsy samples were exposed to repeated PBS washes prior to laser microdissection to remove blood-derived proteins, and glomerular dissectants were analyzed by MS. The depth of proteomic analysis was evaluated by dynamic range of identified proteins and detection of low-abundance proteins. Results: Two times PBS washes of frozen sections effectively eliminated blood-derived pro- teins in laser-microdissected glomeruli and gave an increased number of identified proteins. Analysis of glomeruli from single specimens by a linear ion trap-Orbitrap mass analyzer gen- erated nonredundant, high-confidence datasets of more than 400 identified proteins with high reproducibility, which attained to a considerable depth of the glomerulus proteome as revealed by a wide dynamic range and identification of low-abundance proteins. Conclusions and clinical relevance: Implementation of washing of frozen section with PBS successfully removed blood-derived proteins and resulted in an in-depth proteomic analysis of laser-microdissected glomeruli, suggesting applicability to clinical study. Keywords: Biopsy specimen / Frozen tissue / Human kidney glomerulus / Laser microdissection / LC-MS/MS Received: March 13, 2012 Revised: May 26, 2012 Accepted: May 30, 2012 The glomerulus is the site of plasma filtration and production of primary urine in the kidney. The structure not only plays a Correspondence Dr. Yutaka Yoshida, Department of Structural Pathology, Institute of Nephrology, Graduate School of Medical and Dental Sciences, Niigata University, 1–757 Asahimachi-dori, Chuo-ku, Niigata 951–8510, Japan E-mail: yyoshi@med.niigata-u.ac.jp Fax: +81-25-227-0768 Abbreviations: emPAI, exponentially modified protein abundance index; LMD, laser microdissection; NSAF, normalized spectral abundance factor pivotal role in ultrafiltration of plasma into urine, but also the locus of kidney diseases progressing to chronic renal failure. The diagnosis and treatment of glomerular diseases are now based on clinical manifestations, urinary protein excretion level, and renal pathology of needle biopsy specimens. The cellular and matrix architecture of glomeruli of biopsy speci- mens have been mapped in detail, providing the basis of diag- nosis, classification, and decision of clinical treatment. In con- trast to these detailed morphological changes in the glomeru- lus, the molecular composition of the glomerulus and how it changes with the progress of the diseases are still obscure. C  2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.clinical.proteomics-journal.com