Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS

Abstract

Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77%-92% within replicates and the majority of these repeated proteins (70%) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

Towards reproducible MRM based biomarker discovery using dried blood spots

“…On the other hand, the hypothesis-driven targeted approach, known as multiple reaction monitoring (MRM; also known as selective reaction monitoring), provides for sensitive and robust quantification of pre-selected proteins67. Both approaches have been widely used for quantitative analysis of proteins in various complex sample matrices including Dried Blood Spots (DBS) and serum89101112.…”

“…The use of DBS sampling in the clinical environment first came to prominence with the screening of new-borns for the metabolic disease phenylketonuria24 and is now utilized in screening for a wide range of other metabolic disorders. Although DBS sampling has predominantly been used in metabolite-based clinical diagnostics2526, there is an emerging interest in the use of this alternative biological source in proteomics for biomarker discovery89102728; particularly for diseases such as psychiatric disorders in which patient recruitment is notoriously difficult and expensive. Despite the cost, collection, shipment and storage advantages, the complexity of DBS samples (including cellular components) represents a challenge for proteomics investigations.…”

Water-Soluble Dried Blood Spot in Protein Analysis: A Proof-of-Concept Study

“…Sample preparation performed after the trypsinization step was solely assessed, which is in accordance with many of the previously published articles for sample preparation of protein from DBS (spotted on traditional DBS materials). 10,12,15,16 The methods evaluated initially were SPE and ultrafiltration.…”

Thermal Inactivation of Enzymes and Pathogens in Biosamples for MS Analysis

“…Protocols for untargeted [51][52][53] and targeted [54,55] proteomics have been adapted to DBS samples. Quantitative extraction of large peptides and proteins from DBS samples prior to digestion can be a challenge, and often direct digestion can be a better alternative [55,56].…”