title: Analysis of low-abundance, low-molecular-weight serum proteins using mass spectrometry.
abstract: To detect diseases early in the general population, new diagnostic approaches are needed that have adequate sensitivity and specificity. Recent studies have used mass spectrometry to identify a serum proteomic pattern for breast and ovarian cancer. Serum contains 60-80 mg/mL protein, but 57-71% of this is serum albumin, and 8-26% are gamma-globulins. These large proteins must be depleted before smaller, less-abundant proteins can be detected using mass spectrometry, but because serum albumin is known to act as a carrier for smaller proteins, removal of these molecules using columns or filtration may result in the loss of molecules of interest. The objective of this study was to develop a reproducible method to deplete serum samples of high-abundance proteins in order to analyze the less-abundant proteins present in serum. We used organic solvents to precipitate the large proteins out of solution. We also predicted that this would cause many smaller proteins to dissociate from their carrier molecules, allowing for detection of a larger number of peptides and small proteins. These treated samples were analyzed using capillary liquid chromatography coupled with electrospray ionization mass spectrometry. Analysis demonstrated reproducible results. Acetonitrile treatment clearly released many carrier-bound molecular species and was superior to ultrafiltration alone for serum proteomic analysis.
authors.affiliation: Department of Chemistry and Biochemistry, Brigham Young University, Provo, UT 84602, USA.
authors.forename: Karen
authors.initials: K
authors.lastname: Merrell
authors.forename: Katie
authors.initials: K
authors.lastname: Southwick
authors.forename: Steven W
authors.initials: SW
authors.lastname: Graves
authors.forename: M Sean
authors.initials: MS
authors.lastname: Esplin
authors.forename: Nathan E
authors.initials: NE
authors.lastname: Lewis
authors.forename: Craig D
authors.initials: CD
authors.lastname: Thulin
mesh_terms: D000097:Acetonitriles; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry*; D002623:Chemistry Techniques, Analytical; D002853:Chromatography, Liquid; D006801:Humans; D013058:Mass Spectrometry; D014462:Ultrafiltration
chemical_list: D000097:Acetonitriles; D001798:Blood Proteins; C032159:acetonitrile