Providing an analytical platform for state-of-the-art absolute apolipoprotein quantification in human blood plasma using prm-PASEF

Introduction

With proteomics making a move into precision medicine, there is an increasing pressure on quantitative data quality in studies of blood profiles. Despite the endeavors to develop new data-analysis pipelines, options for improving quantification results early on and prior to the sample preparation are limited. Heavy labelled protein standards are promising solution to this challenge with potential to ensure precise quantification when combined with targeted mass spectrometry. Apolipoproteins are proteins that bind to lipids to form lipoproteins. They have broad diagnostic importance in cardiovascular risk assessment, identification of lipoprotein abnormalities, monitoring treatment efficacy and genetic disorders. We utilized prm-PASEF a powerful targeted mass spectrometry technique to develop a reference target assay for absolute quantitation of apolipoproteins in human blood plasma.

Methods

The ApoEdge panel (ProteomEdge, Sweden), consisting of heavy Lys and Arg 13C and 15N quantitative recombinant protein standards (qRePS) covering all 18 human apolipoproteins, was either digested and analyzed directly or spiked-in to non-depleted plasma, digested and used for absolute quantification. Tryptic digests were loaded onto 25cm C18 column (75μm, 1.6μm, Aurora, IonOpticks) using nanoElute HPLC (Bruker) coupled to timsTOF HT (Bruker) via a CaptiveSpray source (Bruker) using a 35-min ACN gradient. Target method generation was done by analyzing standards using dia-PASEF, followed by library and panel generation in SpectroDive (v11, Biognosys). Generated prm-PASEF panel had a retention time tolerance set to 2 min and ion mobility tolerance set to 0.05 1/K0. Subsequent targeted data processing was done in SpectroDive.

Results

In the presented study we developed a prm-PASEF assay for analysis of Apolipoproteins in human plasma background. Targeted quantitation was based on 19 qRePS spiked into the sample of interest, which allowed determination of absolute concentrations. Assay development was done by analyzing pure labelled samples without background using dia-PASEF for library generation. In total, 225 peptides from all 18 panel proteins were identified using a 35-min gradient. Panel refinement based on initial measurements done in plasma background resulted in a final panel of 114 targets to be measured. In prm-PASEF, several precursors could be selected within one IMS scan and consequently fragmented in the collision cell. For the developed assay up to 3 precursors were scheduled per TIMS frame. Data processing of 8 replicates revealed excellent reproducibility with a median coefficient of variation of 7% for peptides spiked in complex plasma background. We further estimated the concentration of the endogenous proteins by single-point calibration with the reference spike-in standard. In the next step, we will further expand the assay to 175 clinically relevant proteins.