Faster, More Efficient Proteomic Analysis
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The field of Liquid Chromatography-Mass Spectrometry (LC-MS) has seen advances which have increased the technique’s utility for proteomic analysis. One persistent limitation has been the speed and efficiency of analysis. A recent paper identified that only 16% of peptides eluting from an LC gradient were targeted by subsequent MS analysis. The authors showed that the parallel accumulation-serial fragmentation (PASEF) method, which employs trapped ion mobility spectrometry allied to an orthogonal quadrupole time-of-flight mass spectrometer (TIMS-TOF) was able to overcome these limitations.
Bruker Daltonics, whose researchers co-authored the work, have now unveiled the timsTOF Pro, an analyser which utilises this new technology. We spoke to Dr Gary Kruppa, Vice President Proteomics at Bruker Daltonics, about the technique and its potential for advancing proteomic analysis. Dr Kruppa joined Bruker in 1991 as a software and applications developer for mass spectrometry. Becoming Vice President of Proteomics in 2016, Dr. Kruppa now drives market and applications development for Bruker's innovative solutions for research proteomics.
Ruairi Mackenzie (RM): The PASEF method is enabled by the combination of trapped ion mobility spectrometry (TIMS) coupled with Time-of-Flight (TOF) mass spectrometry. Can you explain how these two techniques work?
Dr Gary Kruppa (GK): Trapped Ion Mobility Spectrometry (TIMS) is a gas-phase separation technique, which resolves sample complexity with an added dimension of separation in addition to HPLC and mass spectrometry, increasing peak capacity and confidence in compound characterization. As importantly, the TIMS analyser is a trapping device that accumulates, traps, and focuses ions into peaks that elute over a few milliseconds. Time of flight mass spectrometry (TOF-MS) is an approach to capturing a broad molecular weight range of signals which operates at very high scan rates. The TIMS technology allows a TOF-MS to run at much higher speeds, and together with the additional dimension of separation, unravels the complexity of samples and unlocks insights that remain hidden in standard mass spectrometry.
RM: What are the advantages of bringing the TIMS and TOF techniques together?
GK: Combining the ion focusing effect from TIMS with ultra high resolution and high-speed TOF technology enables optimal performance. TOF mass spectrometers acquire mass spectra at a very high frequency, but historically, the data acquisition speed has been limited by the need to average many scans to get a good resulting mass spectrum from a continuous ion beam. TIMS further enhances performance by trapping and focusing ions according to their mobilities, allowing very rapid acquisition of high quality spectra. This enables the discovery of low level biologically relevant proteins which, currently, non-TIMS MS systems cannot detect. The overall speed advantage is unparalleled compared to other techniques combined with mass spectrometry, such as drift tube ion mobility methods.
RM: Could you outline how PASEF technology works and how it improves on other techniques?
GK: The TIMS separation in the PASEF method focuses ions in time and space according to their mobility, resulting in high density ion packets eluting over just several milliseconds with a total TIMS scan time of just 100 ms for shotgun proteomics experiments. For PASEF MS/MS scans, the mass selection window is synchronized with the elution times of the specific ion packets, allowing it to fragment >10 selected precursors during the 100 ms TIMS scan. This combination of technologies allows faster acquisition of data-dependent MS/MS spectra, with improved sensitivity and no compromise in spectral quality. TIMS-powered PASEF technology enables sequencing speeds of >100 Hz without compromising sensitivity or resolution. With PASEF, parallel accumulation of ions occurs in the first TIMS section, while the TIMS-separated ions are released from a second TIMS analyser section, resulting in a duty cycle near 100%.
RM: How do you see PASEF impacting the field of proteomic analysis?
GK: The timsTOF Pro powered by PASEF will dig deeper into the proteome. With the PASEF method, the MS/MS spectra quality of the low abundant peptides can be increased by selecting them several times. Peptide mixtures are still extremely complex when analysed in two dimensions (retention time and m/z) therefore, by adding another dimension, scientists in the field of shotgun proteomics do not have to choose a trade-off between resolution, scan speed and sensitivity. This new technology has the potential to revolutionize several areas in proteomics: clinical research where speed and robustness are key for running large sample cohorts; applications requiring increased sensitivity; and for quantitation experiments using isobaric tags, where the additional separation using trapped ion mobility can at least partially remove interference from co-fragmenting species that cause the so-called ratio compression effect that leads to unacceptable errors.
Dr Kruppa was speaking to Ruairi Mackenzie, Science Writer for Technology Networks.