title: Analysis of albumin-associated peptides and proteins from ovarian cancer patients.
abstract: BACKGROUND
  Albumin binds low-molecular-weight molecules, including proteins and peptides, which then acquire its longer half-life, thereby protecting the bound species from kidney clearance. We developed an experimental method to isolate albumin in its native state and to then identify [mass spectrometry (MS) sequencing] the corresponding bound low-molecular-weight molecules. We used this method to analyze pooled sera from a human disease study set (high-risk persons without cancer, n = 40; stage I ovarian cancer, n = 30; stage III ovarian cancer, n = 40) to demonstrate the feasibility of this approach as a discovery method.
  
  
  METHODS
  Albumin was isolated by solid-phase affinity capture under native binding and washing conditions. Captured albumin-associated proteins and peptides were separated by gel electrophoresis and subjected to iterative MS sequencing by microcapillary reversed-phase tandem MS. Selected albumin-bound protein fragments were confirmed in human sera by Western blotting and immunocompetition.
  
  
  RESULTS
  In total, 1208 individual protein sequences were predicted from all 3 pools. The predicted sequences were largely fragments derived from proteins with diverse biological functions. More than one third of these fragments were identified by multiple peptide sequences, and more than one half of the identified species were in vivo cleavage products of parent proteins. An estimated 700 serum peptides or proteins were predicted that had not been reported in previous serum databases. Several proteolytic fragments of larger molecules that may be cancer-related were confirmed immunologically in blood by Western blotting and peptide immunocompetition. BRCA2, a 390-kDa low-abundance nuclear protein linked to cancer susceptibility, was represented in sera as a series of specific fragments bound to albumin.
  
  
  CONCLUSIONS
  Carrier-protein harvesting provides a rich source of candidate peptides and proteins with potential diverse tissue and cellular origins that may reflect important disease-related information.
authors.affiliation: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Lowenthal
authors.forename: Arpita I
authors.initials: AI
authors.lastname: Mehta
authors.forename: Kristina
authors.initials: K
authors.lastname: Frogale
authors.forename: Russell W
authors.initials: RW
authors.lastname: Bandle
authors.forename: Robyn P
authors.initials: RP
authors.lastname: Araujo
authors.forename: Brian L
authors.initials: BL
authors.lastname: Hood
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.forename: Paul
authors.initials: P
authors.lastname: Goldsmith
authors.forename: David
authors.initials: D
authors.lastname: Fishman
authors.forename: Emanuel F
authors.initials: EF
authors.lastname: Petricoin
authors.forename: Lance A
authors.initials: LA
authors.lastname: Liotta
mesh_terms: D000418:Albumins / Q000737:chemistry*; D000595:Amino Acid Sequence; D024682:BRCA2 Protein / Q000097:blood / Q000737:chemistry; D015153:Blotting, Western; D005240:Feasibility Studies; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009367:Neoplasm Staging; D010051:Ovarian Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000235:genetics; D010446:Peptide Fragments / Q000097:blood / Q000737:chemistry; D010455:Peptides / Q000097:blood / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein
chemical_list: D000418:Albumins; D024682:BRCA2 Protein; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins