timsTOF Pro and PASEF: Multiplying Sequencing Speed and Sensitivity in Proteomics
Applications of proteomics to cell biology and biomedical research require further developments of mass spectrometry (MS) technology to overcome long-standing limitations in speed, sensitivity and robustness. Trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer has shown much promise in this regard. Here, we further show the integration of ‘Parallel Accumulation followed by Serial Fragmentation’ (PASEF, Meier et al., J Proteome Res 2015).
In this Webinar, we will explain the fundamentals of this technology and give examples of how the timsTOF Pro was applied for high sensitivity and high speed label-free proteome quantification. We will furthermore discuss factors for the success of these experiments, starting from sample preparation and liquid chromatography to method development and data analysis with MaxQuant.
Ion mobility adds a dimension to LC-MS based shotgun proteomics which potentially can boost proteome coverage, quantification accuracy and dynamic range. Required for this is suitable software that extracts the information contained in the four-dimensional data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification in LC-IMS-MS/MS shotgun proteomics data. Here we apply it to Bruker timsTOF Pro data. Application to benchmark datasets show unprecedented identification depth in shingle shot HeLa runs and precise ratio quantification in datasets with known ground truth.