title: A rapid method for cross-species quantitation of apolipoproteins A1, B48 and B100 in plasma by ultra-performance liquid chromatography/tandem mass spectrometry.
abstract: Apolipoprotein B100 (apoB100) and apolipoprotein A1 (apoA1) are the primary protein components of low density lipoprotein (LDL) and high density lipoprotein (HDL) particles, respectively, and plasma levels of these proteins are associated with risks of cardiovascular disease. Existing apoB100 quantitation methods for animal models have been limited to affinity capture techniques such as enzyme-linked immunosorbent assay (ELISA) and Western blot which require specialized reagents for each species and in many cases are not readily available. Here we demonstrate a single translatable ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) assay that is fast and robust and can be used to measure apolipoprotein concentrations in plasma for six species. When possible, peptide sequences that are conserved across species were identified for this assay. The sample preparation is limited and can be carried out in 96-well microtiter plates and thus allows for multiplexed preparation of samples for analysis of large numbers of samples in a short time frame when combined with UPLC/MS/MS. Separation and quantitation of the tryptic peptides is carried out at 700 μL/min using a 1.7 µm core shell C18 column (2.1 × 50 mm). The chromatography is designed for the analysis of over 100 samples per day, and the UPLC run is less than 10 min. This assay is capable of supporting cardiovascular research by providing a single assay to measure critical biomarkers across multiple species without the need for antibodies, and does so in a high-throughput manner.
authors.affiliation: Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065, USA. michael_lassman@merck.com
authors.forename: Michael E
authors.initials: ME
authors.lastname: Lassman
authors.forename: Theresa M
authors.initials: TM
authors.lastname: McLaughlin
authors.forename: Elizabeth P
authors.initials: EP
authors.lastname: Somers
authors.forename: Alice C
authors.initials: AC
authors.lastname: Stefanni
authors.forename: Zhu
authors.initials: Z
authors.lastname: Chen
authors.forename: Beth Ann
authors.initials: BA
authors.lastname: Murphy
authors.forename: Kathleen K
authors.initials: KK
authors.lastname: Bierilo
authors.forename: Amy M
authors.initials: AM
authors.lastname: Flattery
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Wong
authors.forename: Jose M
authors.initials: JM
authors.lastname: Castro-Perez
authors.forename: Brian K
authors.initials: BK
authors.lastname: Hubbard
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Roddy
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016632:Apolipoprotein A-I / Q000097:blood; D053299:Apolipoprotein B-100 / Q000097:blood* / Q000235:genetics; D053283:Apolipoprotein B-48 / Q000097:blood* / Q000235:genetics; D002318:Cardiovascular Diseases / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003198:Computer Simulation; D006224:Cricetinae; D004285:Dogs; D055785:Gene Knockdown Techniques; D006801:Humans; D016014:Linear Models; D008253:Macaca mulatta; D051379:Mice; D010446:Peptide Fragments / Q000032:analysis; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D013045:Species Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D016632:Apolipoprotein A-I; D053299:Apolipoprotein B-100; D053283:Apolipoprotein B-48; D010446:Peptide Fragments; D034741:RNA, Small Interfering; D014357:Trypsin