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Selective washing of dried blood spots provides enhanced coverage and quantification of the blood proteome.

Natasha Lucas; Cameron Hill; Elisabeth Karsten; Rosalee McMahon; Ben Herbert

The microsampling of blood in the form of dried blood spots has several advantages: the convenience of sample collection (small volume and no special equipment or personnel required); the ease of storage and transport; the accessibility of at-home and remote community sampling; and the application of these to longitudinal studies. However, analysis of blood for proteomics using mass spectrometry has always been limited due to the very wide dynamic range. This is most often overcome by utilising multiple offline and online fractionation which can achieve improved coverage. These approaches can produce thousands of protein identifications however, they are not well suited to scale-up for large number of samples.  Using small volume (30 µL) volumetric absorptive microsampling (VAMS) devices we have developed a series of methods to fractionate samples via the use of various wash solutions and in-tip digests. These methods have been found to successfully decrease the dynamic range of dried whole blood and enhance detection of the blood proteome.

We present here various wash buffer combinations (salts, organic solvents, acids, detergents) and demonstrate (using DIA-MS) that we can quantify greater than 3,500 proteins from whole blood and 1,000 proteins from plasma. Each wash produces a distinct protein profile and hence can be used to target specific protein and/or peptide populations. We also demonstrate that our methods enable extraction using solutions typically incompatible with mass spectrometry analysis (i.e. CHAPS) to produce unique and comprehensive datasets without compromising data quality. The flexibility of these methods enables multiple extractions from a single tip for multiplexing of different analyses (i.e. extraction for immunochemistry assay followed by extraction and digestion for mass spectrometry) and they are applicable to a wide range of sample types including whole blood, plasma, peripheral blood mononuclear cells (PBMCs), red blood cell pellets, saliva, and cell lines.

These methods and results outline a new approach to sample preparation that can help increase protein identifications from small volumes, complex protein solutions, and samples containing SDS or CHAPS. Overall, the ability to get excellent coverage and reproducible protein extraction from VAMS devices using fractionation will further enable their use in protein biomarker studies.

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