title: A differential protein solubility approach for the depletion of highly abundant proteins in plasma using ammonium sulfate.
abstract: Depletion of highly abundant proteins is an approved step in blood plasma analysis by mass spectrometry (MS). In this study, we explored a precipitation and differential protein solubility approach as a fractionation strategy for abundant protein removal from plasma. Total proteins from plasma were precipitated with 90% saturated ammonium sulfate, followed by differential solubilization in 55% and 35% saturated ammonium sulfate solutions. Using a four hour liquid chromatography (LC) gradient and an LTQ-Orbitrap XL mass spectrometer, a total of 167 and 224 proteins were identified from the 55% and 35% ammonium sulfate fractions, whereas 235 proteins were found in the remaining protein fractions with at least two unique peptides. SDS-PAGE and exclusive total spectrum counts from LC-MS/MS analyses clearly showed that majority of the abundant plasma proteins were solubilized in 55% and 35% ammonium sulfate solutions, indicating that the remaining protein fraction is of potential interest for identification of less abundant plasma proteins. Serum albumin, serotransferrin, alpha-1-antitrypsin and transthyretin were the abundant proteins that were highly enriched in 55% ammonium sulfate fractions. Immunoglobulins, complement system proteins, and apolipoproteins were among other abundant plasma proteins that were enriched in 35% ammonium sulfate fractions. In the remaining protein fractions a total of 40 unique proteins were identified of which, 32 proteins were identified with at least 10 exclusive spectrum counts. According to PeptideAtlas, 9 of these 32 proteins were estimated to be present at low μg ml(-1) (0.12-1.9 μg ml(-1)) concentrations in the plasma, and 17 at low ng ml(-1) (0.1-55 ng ml(-1)) range.
authors.affiliation: Department of Biosciences, University of Oslo, Oslo, Norway. r.c.bollineni@biotek.uio.no and Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
authors.forename: Ravi Chand
authors.initials: RC
authors.lastname: Bollineni
authors.affiliation: Centre for Molecular Medicine Norway (NCMM), University of Oslo and Oslo University Hospitals, Norway.
authors.forename: Ingrid J
authors.initials: IJ
authors.lastname: Guldvik
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden.
authors.forename: Henrik
authors.initials: H
authors.lastname: Grönberg
authors.affiliation: Department of Medical Epidemiology and Biostatistics, Karolinska Institute, Stockholm, Sweden.
authors.forename: Fredrik
authors.initials: F
authors.lastname: Wiklund
authors.affiliation: Centre for Molecular Medicine Norway (NCMM), University of Oslo and Oslo University Hospitals, Norway and Department of Cancer Prevention, Oslo University Hospitals, Oslo, Norway.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Mills
authors.affiliation: Department of Biosciences, University of Oslo, Oslo, Norway. r.c.bollineni@biotek.uio.no and Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway.
authors.forename: Bernd
authors.initials: B
authors.lastname: Thiede
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry*; D001774:Blood Chemical Analysis / Q000379:methods*; D001798:Blood Proteins / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D012995:Solubility
chemical_list: D001798:Blood Proteins; D000645:Ammonium Sulfate