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, 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.

Keywords: Collisional Cross Section; HPLC; Label-free quantification; Mass Spectrometry; PASEF; Protein Identification; Quantification; Sequencing MS; TIMS; Tandem Mass Spectrometry; ion mobility.

Graphical abstract

Fig. 1.

Online Parallel Accumulation - Serial Fragmentation (PASEF) with the timsTOF Pro. A, Peptides eluting from the chromatographic column are ionized and enter the mass spectrometer through a glass capillary. B, In the dual TIMS analyzer, the first TIMS section traps and stores ion packets, and the second resolves them by mobility. C, D, Ion mobility separated ions are released sequentially from the second TIMS analyzer as a function of decreasing electrical field strength and yield mobility-resolved mass spectra. E, In PASEF MS/MS scans, the TIMS analyzer and the quadrupole are synchronized and the quadrupole isolation window switches within sub-milliseconds between mobility resolved precursor ions of different m/z. F, This yields multiple ion mobility resolved MS/MS spectra from a single TIMS scan, and ensures that multiple trapped precursor ion species are used for fragmentation. Non mobility-resolved MS and MS/MS spectra are projected onto the right axes in (E) and (F) for comparison.

Fig. 2.

Real-time PASEF precursor selection in three dimensions. Heat-map visualization of ion mobility resolved peptide ions at a single time point in an LC-TIMS-MS analysis of a HeLa digest. Connected lines indicate the m/z and mobility positions of all precursor ions selected for fragmentation in the following TIMS-PASEF scans (color-coded).

Fig. 3.

Trapped ion mobility separation of peptide precursor ions. A, The two nearly isobaric peptide ions A and B were distinguished by their ion mobility and selected separately for fragmentation by the PASEF scheduling algorithm in an LC-TIMS-MS experiment of a HeLa digest. B, Zoomed view into the precursor m/z range. Non mobility-resolved MS spectra are projected onto the lower axis for comparison. The corresponding MS/MS spectra are shown in supplemental Fig. S1.

Fig. 4.

Single run analyses of a HeLa digest. A, Number of selected precursor ions per PASEF scan with different TIMS ramp times in 120 min runs of 200 ng HeLa digests. B, Cumulative number of PASEF MS/MS spectra as a function of retention time for 50 ms and 100 ms TIMS ramps. The dashed line indicates the theoretical number of MS/MS spectra for a constant acquisition rate of 100 Hz starting at a retention time of 7.5 min. C, Number of repeated sequencing events for precursors with different ramp times. D, Number of unique precursor ions detected with different TIMS settings. E, Average number of sequence-unique peptides identified in single runs (n = 4) with different TIMS settings. F, Average number of protein group identifications in single runs (n = 4) with different TIMS settings.

Fig. 5.

Label-free proteome quantification. A, Pearson correlation of protein intensities in two replicate injections of a HeLa digest. B, Coefficients of variation (CVs) for protein quantities in four replicates (n = 5,811). C, Number of proteins quantified in N out of four replicates. D, Label-free quantification benchmark with whole-cell HeLa and E. coli digests mixed in 1:1 and 1:5 ratios (wt:wt). The scatterplot shows the median fold-change of 5407 human and 728 E. coli proteins in quintuplicate single runs.

Fig. 6.

Rapid and sensitive HeLa proteome measurements. A, Average number of protein groups identified and quantified with a CV <20% in 60 min single runs (n = 3). B, Average number of protein groups identified and quantified with a CV <20% in 30 min single runs (n = 3). C, Number of protein groups identified in sixteen replicate injections with the 5.6 min gradient.

Fig. 7.

Large-scale and high-precision CCS measurements. A, Pearson correlation of peptide ion mobilities in two replicate injections of a HeLa digest (100 ms TIMS ramps). B, Relative deviations of ^TIMSCCS values of all individual peptides from their mean of quadruplicate LC-MS runs (n = 144,363; 1,186 out of range). C, Pearson correlation of measured ^TIMSCCS values in two injections of a HeLa digest with different TIMS ramp times (100 and 200 ms TIMS ramps). D, Density distribution of 129,110 ^TIMSCCS values from human tryptic peptide ions as a function of m/z. The main populations are annotated with their respective charge states.