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.

Automated Multiplex LC-MS/MS Assay for Quantifying Serum Apolipoproteins A-I, B, C-I, C-II, C-III, and E with Qualitative Apolipoprotein E Phenotyping

“…We selected 2 signature peptides per apolipoprotein on the basis of our previous work, published literature (17)(18)(19)30 ), and signal intensities during LC-MS/MS analyses of tryptic peptide candidates. The peptide with the most reproducible results was selected as quantification peptide, whereas the other peptide was used for confirmation (23 ).…”

A sensitive and robust lc-ms/ms method with monolithic column and electrospray ionization for the quantitation of efavirenz in human plasma: application to a bioequivalence study

“…Recent approaches applied to increase overall productivity in high-throughput applications include the use of simultaneous positive and negative electrospray ionization 41 and the use of ultra performance liquid chromatography (UPLC) for shorter analysis times. 42 In this study LC-MS/MS with liquid-liquid extraction method for the quantification of efavirenz demonstrated good precision, accuracy and linearity, with a short analytical time, reducing the total analysis time. Several LC-MS/MS methods described in the literature were developed for simultaneous quantification of various anti-HIV agents with a long analytical time.…”

A proteomic approach combining MS and bioinformatic analysis for the detection and identification of biomarkers of administration of exogenous human growth hormone in humans

“…Further validation will be required before LRG can be added to the collection of rhGH-related biomarkers for testing purposes. This should be performed using a larger sample set with a high-throughput LC-MS/MS and MRM-based assay in conjunction with stable isotope-labelled peptides [37] or possibly using immunoassay-based techniques [38]. This would allow quantitative data to be generated through the analysis of samples from the original and additional rhGH administration studies.…”