Abstract
Discovery of protein biomarkers in clinical samples necessitates significant prefractionation prior to liquid chromatography–mass spectrometry (LC–MS) analysis. Integrating traveling wave ion mobility spectrometry (TWIMS) enables in-line gas phase separation which when coupled with nanoflow liquid chromatography and data independent acquisition tandem mass spectrometry, confers significant advantages to the discovery of protein biomarkers by improving separation and inherent sensitivity. Incorporation of TWIMS leads to a packet of concentrated ions which ultimately provides a significant improvement in sensitivity. As a consequence of ion packeting, when present at high concentrations, accurate quantitation of proteins can be affected due to detector saturation effects. Human plasma was analyzed in triplicate using liquid-chromatography data independent acquisition mass spectrometry (LC-DIA-MS) and using liquid-chromatography ion-mobility data independent acquisition mass spectrometry (LC-IM-DIA-MS). The inclusion of TWIMS was assessed for the effect on sample throughput, data integrity, confidence of protein and peptide identification, and dynamic range. The number of identified proteins is significantly increased by an average of 84% while both the precursor and product mass accuracies are maintained between the modalities. Sample dynamic range is also maintained while quantitation is achieved for all but the most abundant proteins by incorporating a novel data interpretation method that allows accurate quantitation to occur. This additional separation is all achieved within a workflow with no discernible deleterious effect on throughput. Consequently, TWIMS greatly enhances proteome coverage and can be reliably used for quantification when using an alternative product ion quantification strategy. Using TWIMS in biomarker discovery in human plasma is thus recommended.
Highlights
Biomarkers are molecules which alter as a consequence of disease etiology and progression and reflect the status of the disease.[1]
In order to investigate a greater proportion of the plasma proteome, prefractionation strategies including 2D-reversed phase (RP)-RP,[6] ion exchange (IEX)-RP,[7,8] strong cation exchange (SCX)-RP,[8,9] liquid electrophoresis,[10] and 1D-gel LC−MS11 are often utilized, and these lead to successful levels of separation but with significant decreases in throughput, making large clinical studies unviable.[12]
Data shown in this paper demonstrate that the challenges in quantitation when incorporating traveling wave ion mobility spectrometry (TWIMS), which are associated with signal saturation, can be overcome using an alternative, MS2-based, label-free, absolute quantitation method
Summary
Biomarkers are molecules which alter as a consequence of disease etiology and progression and reflect the status of the disease.[1]. In order to investigate a greater proportion of the plasma proteome, prefractionation strategies including 2D-reversed phase (RP)-RP,[6] ion exchange (IEX)-RP,[7,8] strong cation exchange (SCX)-RP,[8,9] liquid electrophoresis,[10] and 1D-gel LC−MS11 are often utilized, and these lead to successful levels of separation but with significant decreases in throughput, making large clinical studies unviable.[12] Incorporating traveling wave ion mobility orthogonally to the time-of-flight (TOF) analyzer (oa-TOF)[13] enables a gas phase separation which, because of its orthogonality, provides additional separation at no additional cost to throughput.[14] the incorporation of traveling wave ion mobility spectrometry (TWIMS) can be used to replace prefractionation strategies and maintain a reasonable throughput or used within a prefractionation pipeline to significantly improve the overall coverage. Ion mobility (IM) separation is on the millisecond time scale, nesting within the time scale of nano-LC separation (seconds) and TOF-MS acquisition (∼100 μs), allowing multiple mass spectra to be taken of each ion mobility separation[16] and providing an additional degree of peak capacity.[17]
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