A centrifugal ultrafiltration strategy for isolating the low-molecular weight (&lt;= 25 K) component of human plasma proteome

Greening, DW; Simpson, RJ

A centrifugal ultrafiltration strategy for isolating the low-molecular weight (<= 25 K) component of human plasma proteome

Author(s): Greening, DW; Simpson, RJ;
Details: Publication Year 2010-01-03,Volume 73,Issue #3,Page 637-648
Journal Title: JOURNAL OF PROTEOMICS
Publication Type: Journal Article
Abstract: The low-molecular weight fraction (IMF) of the human plasma proteome is an invaluable source of biological information, especially in the context of identifying plasma-based biomarkers of disease. In this study, a separation and enrichment strategy based on centrifugal ultrafiltration was developed for the IMF (i.e., <= 25 K) of plasma routinely prepared from normal, healthy volunteers. Four commercially-available filter membranes of similar nominal molecular weight cut-off (NMWC), but differing membrane chemistries and filter orientations (Microcon (R), Millipore; Centrisart (R), Sartorius; Amicon Ultra (R), Millipore; Vivaspin (R), Sartorius), were evaluated. Of these filtration devices, only the Sartorius Vivaspin (R) tangential membrane, NMWC 20 K was effective in the non-retention of M(r)>50 K, and recovery and enrichment of low-M, components from human plasma. This filter membrane device was further optimized with respect to plasma buffer composition, centrifugal force, duration and temperature. Optimal ultrafiltration conditions were obtained using 100 mu L of normal plasma in 10% acetonitrile, and a centrifugation force of 4000 x 9 for 35 min at 20 degrees C. In this LMF, 44 proteins (from 266 unique peptides) were identified using a combination of 1D-SDS-PAGE/nano-LC-MS/MS and astringent level of identification (FDR <1%). We report the identification of several proteins (e.g., protein KIAA0649 (Q9Y4D3), rheumatoid factor D5, serine protease inhibitor A3, and transmembrane adapter protein PAG) previously not reported in extant high-confidence Human Proteome Organization (HUPO) Plasma Proteome Project datasets. When compared with the low-M, human plasma/serum proteome datasets of Zhou et al. (Electrophoresis, 2004. 25, 1289-98), Gundry et al. (Proteomics Clin. Appl., 2007. 1, 73-88) and Villanueva et al. (Anal Chem, 2004. 76, 1560-70), 64% of our identifications (28 proteins) were novel; these include cofilin-1, PPlase A, and the SH3 domain-binding glutamic acid-rich-like protein 3. In addition to intact proteins, many peptide fragments from high-abundance proteins (e.g., fibrinogen, clusterin, Factor XIIIa, transferrin, kinogen-1, and inter-alpha-trypsin inhibitor), presumably derived by ex vivo proteolysis, were observed. (C) 2009 Elsevier B.V. All rights reserved.
Publisher: ELSEVIER SCIENCE BV
Keywords: LOW-ABUNDANCE PROTEINS; HUMAN TUMOR XENOGRAFT; HUMAN SERUM PROTEOME; MASS-SPECTROMETRY; BLOOD-PLASMA; VASCULAR-PERMEABILITY; HIGH-CONFIDENCE; ALBUMIN; IDENTIFICATION; PEPTIDOME
Publisher's Version: https://doi.org/10.1016/j.jprot.2009.09.013
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Creation Date: 2010-01-03 12:00:00