Simple and Sensitive Analysis for Dried Blood Spot Proteins by Sodium Carbonate Precipitation for Clinical Proteomics

Abstract

Dried blood spots (DBS) are widely used for screening biomolecular profiles, including enzymatic activities. However, detection of minor proteins in DBS by liquid chromatography–mass spectrometry (LC-MS/MS) without pre-enrichment remains challenging because of the coexistence of large quantities of hydrophilic proteins. In this study, we address this problem by developing a simple method using sodium carbonate precipitation (SCP). SCP enriches hydrophobic proteins from DBS, allowing substantial removal of soluble proteins. In combination with SCP, we used quantitative LC-MS/MS proteome analysis in a data-independent acquisition mode (DIA) to enhance the sensitivity and quantification limits of proteome analysis. As a result, identification of 1977 proteins in DBS is possible, including 585 disease-related proteins listed in the Online Mendelian Inheritance in Man.

Non-Lethal Blood Sampling of Fish in the lab and Field With Methods for Dried Blood Plasma Spot Omic Analyses

“…There was an average of 900, 757, 644, and 621 protein identifications made for the WP, PF, WP Disc, and PF Disc sample preparation methods (SPMs), respectively, which offers many possibilities for biomarker discovery and analysis. When compared to WP, the PF, PF Disc, and WP Disc SPMs still performed relatively well considering dried plasma and blood spots generally have lower recoveries and identifications of proteins relative to regular plasma samples ( Chambers et al, 2013 ; Nakajima et al, 2020 ). In order to estimate protein recoveries for SPMs, we calculated the fold change of the SPMs relative to WP.…”

“…Over 90% of proteins detected within the current dataset had negative GRAVY values suggesting that both proteomics sample preparation methods (i.e., Bligh-Dyer extraction or dissolution in AB buffer) select a similar range of mostly hydrophilic proteins regardless of the SPM used. Hydrophobic, native proteins tend to occupy cellular membranes and are not recovered well without specialized sample extraction protocols such as the use of a detergent ( Ludwig et al, 2018 ; Nakajima et al, 2020 ). It was expected that the PF DPS may affect the abundances of hydrophobic proteins considering it is a polymer primarily composed of paraffin waxes and polyolefins which create a non-polar support material, but there were few hydrophobic proteins identified within the dataset ( Miller, 2012 ).…”

Toward proteome‐wide exploration of proteins in dried blood spots using liquid chromatography‐coupled mass spectrometry

“…While the DIA-based approach can be considered an improvement in in-depth DBS proteomics over the DDAbased approach, the depth of proteome coverage is still far from satisfactory. We also attempted to improve DBS proteome analysis with an improved DIA method, that is overlapping windows DIA [48]; we were able to obtain slightly more DBS proteome data using this method, as shown in Table 2 [49]. The depth of DBS proteome analysis was further improved by first fractionating the mass range to five fractions using gas phase fractionation and then applying overlapping window DIA measurements (Table 3).…”

“…When nontarget proteome analysis is intended for use with NBS, it is expected to expand the range of DBS proteins detected by LC-MS/MS analysis with prefractionation to levels equivalent to those achieved with high-throughput analysis in terms of cost and labour. Although few studies have tackled this issue in DBS proteome analysis [49,50], we recently demonstrated that precipitation with sodium carbonate might be useful for this purpose (Table 3) [49].…”

“…This method could be an alternative to antibody assisted enrichment because it enabled us to detect target proteins derived from diseasecausative genes, such as ATP7B, BTK, CYBB, and ADA, without using antibodies [49]. More importantly, 585 proteins registered in OMIM (https://www.omim.org), a database of human genetic mutations and diseases, were observed using this method.…”