Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation

Abstract

The use of protein biomarkers as surrogates for clinical endpoints requires extensive multilevel validation including development of robust and sensitive assays for precise measurement of protein concentration. Multiple reaction monitoring (MRM) is a well-established mass-spectrometric method that can be used for reproducible protein-concentration measurements in biological specimens collected via microsampling. The dried blood spot (DBS) microsampling technique can be performed non-invasively without the expertise of a phlebotomist, and can enhance analyte stability which facilitate the application of this technique in retrospective studies while providing lower storage and shipping costs, because cold-chain logistics can be eliminated. Thus, precise, sensitive, and multiplexed methods for measuring protein concentrations in DBSs can be used for de novo biomarker discovery and for biomarker quantification or verification experiments. To achieve this goal, MRM assays were developed for multiplexed concentration measurement of proteins in DBSs.The lower limit of quantification (LLOQ) was found to have a median total coefficient of variation (CV) of 18% for 245 proteins, whereas the median LLOQ was 5 fmol of peptide injected on column, and the median inter-day CV over 4 days for measuring endogenous protein concentration was 8%. The majority (88%) of the assays displayed parallelism, whereas the peptide standards remained stable throughout the assay workflow and after exposure to multiple freeze-thaw cycles. For 190 proteins, the measured protein concentrations remained stable in DBS stored at ambient laboratory temperature for up to 2 months. Finally, the developed assays were used to measure the concentration ranges for 200 proteins in twenty same sex, same race and age matched individuals.

The clinical potential of prm-PASEF mass spectrometry

“…By focussing the mass spectrometer, the analysis gains sensitivity and, in contrast to shotgun-based discovery methods, enables the observation of the analytes in every sample. Therefore, targeted methods are particularly well suited for measuring larger cohorts and clinical applications [8,14].…”

A high-stringency blueprint of the human proteome

“…Targeted MS methods have been developed, again especially where interferences obfuscate immunoassays 128 or where additional biological context is required 129 . Likewise, volumetric absorptive micro-sampling VAMS blood collection devices (e.g., Mitra devices or dried blood spots) allow patients to mail samples from home to analytical labs to undertake SRM/MRM/PRM assays quantifying CVD risk-associated apolipoproteins 130 or other markers 131 . In summary, consumer-based CVD proteomics-based precision medicine testing services are now coming of age.…”

Evaluation of 92 cardiovascular proteins in dried blood spots collected under field‐conditions

“…Previous studies [7,[12][13][14] have demonstrated the possibility of characterizing protein abundance levels from DBS, both for fewer analytes in large sample sets and for a higher number of proteins from smaller samples sets constructed under laboratory conditions. The recent work by Eshghi and colleagues [12] reports encouraging results: absolute concentrations were determined for over 200 proteins by mass spectrometry. In that study, 50 µL of blood drawn using finger prick technique was pipetted onto spots of the same type of paper cards used here and then five 6 mm punches (one from each spot) was used for analysis.…”