Abstract
Despite the great potential of dried blood spots (DBS) as a source of endogenous proteins for biomarker discovery, the literature relating to nontargeted bottom-up proteomics of DBS is sparse, primarily due to the inherent complexity and very high dynamic range associated with these samples. Here, we present proof-of-concept results in which we have coupled high field asymmetric waveform ion mobility spectrometry (FAIMS) with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for nontargeted bottom-up proteomics of DBS with the aim of addressing these challenges. We, and others, have previously demonstrated the benefits of FAIMS more generally in proteomics including improved signal-to-noise and extended proteome coverage, and the aim of the current work was to extend those benefits specifically to DBS. The DBS samples were either extracted by the more traditional manual "punch and elute" approach or by an automated liquid surface extraction (LESA) approach prior to trypsin digestion. The resulting samples were analyzed by LC-MS/MS and LC-FAIMS-MS/MS analysis. The results show that the total number of proteins identified increased by ∼50% for the punch and elute samples and ∼45% for the LESA samples in the LC-FAIMS-MS/MS analysis. For both the punch and elute samples and the LESA samples, ∼30% of the total proteins identified were observed in both the LC-MS/MS and the LC-FAIMS-MS/MS data sets, illustrating the complementarity of the approaches. Overall, this work demonstrates the benefits of inclusion of FAIMS for nontargeted proteomics of DBS.
Highlights
Proteomics-based studies are useful in the search for new biomarkers.[1]
liquid chromatography (LC)−field asymmetric waveform ion mobility spectrometry (FAIMS)−MS is evaluated for the proteomic analysis of Dried blood spots (DBS) samples using two different sample preparation procedures: (1) punch, elute, and overnight tryptic digest of the DBS, referred to here as punch and elute, and (2) liquid extraction surface analysis (LESA) of DBS combined with a 1 h tryptic digest using the Triversa Nanomate robotic platform from Advion as described by Martin et al.[13]
The samples from the punch and elute procedure were pooled after digestion, as were those from the LESA procedure, to ensure that any differences observed were due to the FAIMS and not differences in extraction or digestion of the proteins
Summary
Proteomics-based studies are useful in the search for new biomarkers.[1]. Blood is a rich source of endogenous proteins and has great potential for proteomics-based biomarker discovery;[2] sampling of blood from large patient cohorts can be both resource-demanding and time-consuming, making the recruitment of patients into these studies difficult. This sampling technique was introduced in 1963 as a means to sampling and storage of whole blood in newborn screening.[3] DBS are easy to obtain and ship, and are well-suited to sampling from large patient cohorts, including in inaccessible regions of the world, without the need for transportation of the patient to the clinic.[4,5] Several articles have described the application of MS for targeted protein analysis of DBS,[6−12] but only two have considered nontargeted protein analysis[13−15] by use of bottom-up proteomics This is surprising given the wide body of research on the human plasma proteome[16,17] but is likely the consequence of the complexity of blood samples and high dynamic range of protein concentrations. These challenges are not peculiar to proteomics-based studies of DBS or other blood derived samples and are typically addressed in proteomics workflows through the use of prefractionation methods such as depletion kits (for removal of high abundance proteins),[18] gel based separation techniques,[19] or strong cation exchange chromatography[20] prior to online liquid chromatography (LC)−tandem mass spectrometry (MS/MS)
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