Online parallel accumulation − serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer

Abstract

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, while mass spectra are acquired in about 100 microseconds with time-of-fight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive due to its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulationserial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 800,000 fragmentation spectra in standard 120 min LC runs are easily achievable, which can be used for near exhaustive precursor selection in complex mixtures or re-sequencing weak precursors. MaxQuant identified more than 6,400 proteins in single run HeLa analyses without matching to a library, and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,900 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

MaxQuant Software for Ion Mobility Enhanced Shotgun Proteomics

“…Ion mobility spectrometry (1-3) (IMS) separates molecules in the gas phase by their collisional cross section (CCS) which is the effective area of a molecule quantifying the likelihood of scattering events with the gas. It can be coupled to mass spectrometry (MS) for which it constitutes a separation dimension in addition to mass over charge (m/z).Together with liquid chromatography (LC) and tandem mass spectrometry (MS/MS) one obtains LC-IMS-MS/MS shotgun proteomics, a promising strategy for the analysis of complex samples (4)(5)(6)(7).Specifically, the object of our studies is the timsTOF Pro instrument (8) which is a time-of-flight (TOF) mass spectrometer utilizing a trapped ion mobility spectrometry (9-11) (TIMS)device operated with the parallel accumulationserial fragmentation (PASEF) scan mode (12).…”

Proteogenomic analysis of granulocyte macrophage colony- stimulating factor autoantibodies in the blood of a patient with autoimmune pulmonary alveolar proteinosis

“…Ions were collected in the TIMS device over 100 ms and MS and MS/ MS data were acquired over an m/z range of 100-1,700. During the collection of MS/MS data, the TIMS cycle was adjusted to 1.1 s and included 1 MS plus 10 PASEF-MS/MS scans, each containing on average 12 MS/MS spectra (>100 Hz) 15,16 .…”

Boric Acid (H3(BO3): Recognize The Molecular Interactions in Solutions

“…Senyawa asam borat memiliki kelarutan yang sedikit jika dilrutkan di dalam alkohol, (139)(140)(141)(142) cukup larut dalam piridina (143) dan akan sangat sedikit larut (144) ketika pelarutnya aseton. (142; 145-148) Kelarutan suatu senyawa dapat dipengaruhi oleh beberapa faktor diantaranya sifat dari zat terlarut (solute) (149)(150)(151)(152)(153) dan pelarut (154) (solvent) (105; 139; 142; 155; 156) dimana zat terlarut yang bersifat polar (214)(215)(216) akan mudah larut dalam solvent (243)(244)(245)(246) yang bersifat polar begitupun sebaliknya. Suhu, konsentrasi, (106; 149; 157-159) pengaruh penambahan ion senama (236)(237) juga sangat berpengaruh terhadap kelarutan (247)(248)(249) suatu senyawa.…”