Project description:Data independent acquisition (DIA or DIA/SWATH ) mass spectrometry has emerged as a primary measurement strategy in the field of quantitative proteomics. diaPASEF is a recent adaptation that leverages trapped ion mobility spectrometry (TIMS) to improve selectivity and increase sensitivity. The complex fragmentation spectra generated by co-isolation of peptides in DIA mode are most typically analyzed with reference to prior knowledge in the form of spectral libraries. The best established method for generating libraries uses data dependent acquisition (DDA) mode, or DIA mode if appropriately deconvoluted, often including offline fractionation to increase depth of coverage,to create spectral libraries. More recently strategies for spectral library generation based on gas phase fractionation (GPF), where a representative sample is injected serially using narrow window DIA methods designed to cover different slices of the precursor space, have been introduced and performed comparably to deep offline fractionation-based libraries for DIA data analysis. Here, we investigated whether an analogous GPF-based library building approach that accounts for the ion mobility (IM) dimension is useful for the analysis of diaPASEF data and can remove the need for offline fractionation. To enable a rapid library development approach for diaPASEF we designed a GPF acquisition scheme covering the majority of multiply charged precursors in the m/z vs 1/K0 space requiring 7 injections of a representative sample and compared this with libraries generated by direct deconvolution-based analysis of diaPASEF data or by deep offline fractionation and ddaPASEF. . We found that the GPF based library outperformed library generation by direct deconvolution of the diaPASEF data, and performed comparably to deep offline fractionation libraries, when analysing diaPASEF data acquired from 200ng of commercial HeLa digest. With the ion mobility integrated GPF scheme we establish a pragmatic approach to rapid and comprehensive library generation for the analysis of diaPASEF data.

Project description:Targeted mass spectrometry (MS) approaches, which are powerful methods for uniquely and confidently quantifying a specific panel of proteins in complex biological samples, play a crucial role in validation and clinical translation of protein biomarkers discovered by global proteomic profiling. Common targeted MS methods, such as multiple reaction monitoring (MRM) and parallel-reaction monitoring (PRM), employ specific mass spectrometric technologies to quantify protein levels by comparing the transitions of specific endogenous (Endo) peptides with those of stable isotope labeled (SIL) peptide counterparts. These methods utilizing amino acid analyzed (AAA) SIL peptides warrants sensitive and precise measurements required for targeted MS assays. Compared to MRM, PRM provides higher experimental throughput by simultaneously acquiring all transitions of the target peptides and thereby compensate for different ion suppression among transitions of a target peptide. However, PRM still suffers different ion suppressions between Endo and SIL peptides due to poor spray stability as the Endo and SIL peptides were monitored at different liquid chromatography (LC) retention times. Here we introduce a new targeted MS method, termed as wideband PRM (WBPRM), designed for high-throughput targeted MS approach. WBPRM employs a wide isolation window for simultaneous fragmentations of both Endo and SIL peptides along with multiplexed single ion monitoring (SIM) scans for enhanced MS sensitivity of target peptides. Compared to PRM, WBPRM was demonstrated to provide increased sensitivity, precision, and accuracy of quantitative measurements of target peptides with increased throughput, allowing more target peptide measurements in a short experiment time. Its adaptability to a MS method provided by the manufacture makes it a facile method to implement for MS based assays, particularly in complex biological samples, where the needs for higher accuracy, sensitivity and efficiency are paramount.

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EVs enriched from plasma by placental proteins

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