The timsTOF Pro offers a combination of two unique technologies, namely a 4th dimension provided by Trapped Ion Mobility Spectrometry (TIMS) to enhance ion separation and sensitivity and Parallel Accumulation Serial Fragmentation (PASEF [1]) to improve ion utilization efficiency and data acquisition speed.
In this application note, we demonstrate the benefits of dia-PASEF technology on the timsTOF Pro platform coupled to an Evosep One for high-throughput in-depth proteome analysis of up to 300 samples per day (SPD). We quantify about 5200 protein groups in only 21- minutes run time. In 4.8-minutes ultra-high throughput runs (300 SPD) we are still able to quantify more than 2000 protein groups and 8500 peptides.
Data-independent acquisition (DIA) facilitates reproducible and accurate protein identification and quantification across large sample cohorts. This is achieved by using isolation of wide quadrupole windows, rather than selecting individual precursors, to ensure that all precursor ions are fragmented in every sample. Ion mobility provides an additional dimension of separation for complex samples, which can also be used for aligning precursor and fragment ions. By making use of reproducible mobility values from the timsTOF Pro, we extend the PASEF principle to DIA resulting in a new acquisition mode, called dia-PASEF [2]. This approach benefits from the sensitivity of PASEF and improves efficiency of ion usage for MS/MS with up to 100% of the ion population being fragmented for MS/MS analysis.
We previously showed the applicability of dia-PASEF for different gradient lengths, with about 6400 protein groups identified in single 30-minute runs from human cell lines [3].
>> Download the Full Application Note as a PDF
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