title: The application of ultra-performance liquid chromatography/tandem mass spectrometry to the detection and quantitation of apolipoproteins in human serum.
abstract: The detection and quantitation of apolipoproteins, important markers for coronary heart disease, in serum by liquid chromatography/tandem mass spectrometry (LC/MS/MS) using multiple reaction monitoring (MRM) is reported. A tryptic digest of depleted human serum was analysed by nanoflow LC/MS/MS at a flow rate of 300 nL/min and several apolipoproteins (Apo), including Apo A1, A2, A4, C1, C2, C3, D, F and M, were successfully identified. The analysis of the same depleted serum digest by ultra-performance (UP)LC/MS/MS operating at 700 microL/min resulted in comparable sensitivity and selectivity to the nanoflow method, but with a dramatic ( approximately 20-fold) reduction in run time. The potential of UPLC/MS/MS for the rapid quantitation of proteins in biological matrices by representative tryptic peptides was further investigated using Apo A1 and its corresponding stable isotopically labelled tryptic AQUA peptide (DYVSQFEGSALGK). A set of serum-based Apo A1 calibrators from a clinical analyser kit were digested without depletion following the addition of the AQUA peptide and analysed using UPLC/MS/MS. A linear calibration curve was generated from peak area ratios to the labelled peptide with a coefficient of correlation of 0.9989. Standard curves were also generated for other apolipoproteins together with Apo B100, Apo E, lecithin cholesterol acyltransferase and albumin, which were also detected in the standards. The concentration of Apo A1 in five fresh undepleted human serum samples and a quality control (QC) sample were determined using both the UPLC/MS/MS method and a clinical analyser. Results were comparable and the quantitative study, involving 80 injections which took hours rather than days to complete, demonstrates the high-throughput potential of UPLC/MS/MS to quantify multiple serum proteins without the need for antibodies, and thus provide an alternative to the use of clinical analysers for serum protein biomarkers.
authors.affiliation: HFL Ltd., Newmarket Road, Fordham, Cambridge CB7 5WW, UK. rkay@hfl.co.uk
authors.forename: Richard G
authors.initials: RG
authors.lastname: Kay
authors.forename: Barbara
authors.initials: B
authors.lastname: Gregory
authors.forename: Philip B
authors.initials: PB
authors.lastname: Grace
authors.forename: Steve
authors.initials: S
authors.lastname: Pleasance
mesh_terms: D001053:Apolipoproteins / Q000097:blood* / Q000737:chemistry*; D001774:Blood Chemical Analysis / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D008832:Microchemistry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D001053:Apolipoproteins