Details

Autor(en) / Beteiligte

• Meier, Florian
• Brunner, Andreas-David
• Koch, Scarlet
• Koch, Heiner
• Lubeck, Markus
• Krause, Michael
• Goedecke, Niels
• Decker, Jens
• Kosinski, Thomas
• Park, Melvin A.
• Bache, Nicolai
• Hoerning, Ole
• Cox, Jürgen
• Räther, Oliver
• Mann, Matthias

Titel

Online Parallel Accumulation–Serial Fragmentation (PASEF) with a Novel Trapped Ion Mobility Mass Spectrometer

Ist Teil von

• Molecular & cellular proteomics, 2018-12, Vol.17 (12), p.2534-2545

Ort / Verlag

United States: Elsevier Inc

Erscheinungsjahr

2018

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• PASEF multiplies the sequencing speed without any loss in sensitivity and is implemented in the timsTOF Pro instrument introduced here. Sequencing speeds above 100 Hz enable single run proteome analysis at a depth of 6000 proteins, making the instrument particularly attractive for rapid and highly sensitive proteomics. Collisional cross sections can be determined with up to 0.1% precision and acquired on a scale of 100,000s, which opens exciting areas for proteomics exploration. [Display omitted] Highlights •Online PASEF achieves greater than 100 MS/MS per second at full sensitivity.•Accurate label-free quantification of over 6000 proteins in 2 h.•High throughput demonstrated on 50 ng digests measured in 5 min.•High-precision determination of 100,000 peptide collisional cross sections. In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial 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 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 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.