GNPS - Ion Mobility-Reframe Drug Library
Ontology highlight
ABSTRACT:
INSTRUMENT(S): timsTOF Pro 2
ORGANISM(S): Synthetic Construct (ncbitaxon:32630)
SUBMITTER: 
Pieter C. Dorrestein
PROVIDER: MSV000098263 | GNPS | Wed Jun 18 18:57:00 BST 2025
REPOSITORIES: GNPS
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Project description:Crude standards of reframe drug mixtures were analyzed on a TimsTOF Pro2 coupled to Agilent LC system for library generation.
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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.
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Project description:Generation of a new library of targeted mass spectrometry assays for accurate protein quantification in triple negative breast cancer (TNBC) tissues. Primary tumor tissue lysates from 105 TNBC patients treated at Masaryk Memorial Cancer Institute (MMCI) in Brno, Czech Republic, were used to generate the spectral library. This project covers raw files from data-dependent acquisition (DDA) – parallel accumulation-serial fragmentation (PASEF) measurements of 12 hydrophilic chromatography (HILIC) fractions of aliquot pool from complete set of 105 samples measured on timsTOF Pro; raw files of 16 individual samples measured in data-independent acquisition (DIA) – PASEF mode and used for hybrid library generation and for demonstrative quantitative DIA data extraction; Pulsar archive generated in Spectronaut 16.0 from 12 DDA-PASEF measurements of HILIC fractions and from 16 data-independent acquisition DIA-PASEF measurements of individual samples. The 16 DIA-PASEF runs of individual samples used for library generation were analyzed using newest versions of Spectronaut (version 18.5) and DIA-NN (version 1.8.1) software tools in library-based setting using the newly generated library as well as in library-free setting showing library-based method to outperform the use of predicted libraries in the terms of identification numbers.
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