title: Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.
abstract: The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens.
authors.affiliation: Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: John B
authors.initials: JB
authors.lastname: Mangrum
authors.affiliation: Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Erika J
authors.initials: EJ
authors.lastname: Martin
authors.affiliation: Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Donald F
authors.initials: DF
authors.lastname: Brophy
authors.affiliation: Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA.|Department of Pharmacotherapy & Outcomes Science, Virginia Commonwealth University, Richmond, VA, USA.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Hawkridge
mesh_terms: D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D056945:Hep G2 Cells; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001798:Blood Proteins; D020543:Proteome
keywords: Plasma proteins; Quantitation; Secretome; Super-SILAC; Technology