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Benchmarking the Q Exactive Hf-X Ms for Shotgun Proteomics

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Benchmarking the Q Exactive Hf-X Ms for Shotgun Proteomics Benchmarking the Q Exactive An Executive Summary HF-X MS for Shotgun Proteomics Hardware and software improvements allow researchers to push the speed and sensitivity limits of protein identification using quadrupole mass spectrometry. Jesper V. Olsen Introduction Professor Shotgun proteomics is a powerful technique for the global analysis of proteins and their post- Novo Nordisk Foundation Center for Protein Research translational modifications. The technique involves the proteolytic digestion of complex proteins University of Copenhagen into shorter peptides, which are subsequently separated by nanoscale liquid chromatography, ionized by electrospray ionization, and directly analyzed in a tandem mass spectrometer (LC-MS/MS), followed by bioinformatic interpretation. A newly introduced instrument, the Thermo Scientific™ Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap™ Mass Spectrometer incorporates advances in source inlet, software algorithms and fragmentation technology, which increase the speed of the analysis and the number of proteins that can be identified in bottom-up proteomics. Using various MS methods and LC gradient lengths, several gains can be obtained by using ultrashort gradients with a farst acquisition method (1). This paper also presents data obtained in more complex experiments, such as phosphoproteomics with tandem isobaric mass tag (TMT10-plex), and data-independent acquisition (DIA) analysis of proteins. Significant Hardware Improvements for Faster Analyses Three advances in the hardware of the Q Exactive HF-X mass spectrom- eter account for the reported improvements in speed and sensitivity (see Figure 1). The first is a redesigned source inlet, which is now similar to the one used in the Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ Mass Spectrometer, with an ion funnel design coupled to a high-capacity transfer tube (HCTT). The second is an optimized ion movement compartment that minimizes ion transfers and allows for much faster, higher- energy collisional dissociation (HCD) fragmentation scanning. The third improvement comes from the addition of new resolution settings, 7,500 (FWHM) at m/z 200 for maximum MS/ MS coverage and 45,000 (FWHM) at m/z 200, which is useful for tandem mass tag (TMT) reporter ion scanning. SPONSORED BY BENCHMARKING THE Q EXACTIVE HF-X MS FOR SHOTGUN PROTEOMICS Q Exactive HF-X MS: novel features To compare the sensitivity Figure 1: Q Exactive HF-X MS: novel features. of the Q Exactive HF-X mass spectrometer to the previous- Optimized ion movements to generation instrument (Thermo minimize ion-transfer times Scientific™ Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ MS), researchers used tryptic digests separated by high- performance liquid chromatog- raphy (HPLC). Monitoring more than 100,000 different peptides, they performed side-by-side analyses using both instruments. A linear regression of the results New source inlet shows that the Q Exactive HF-X ion funnel design mass spectrometer achieves an intensity increase of about 70%, New Orbitrap resolution and on average, up to two-fold settings (7500 & 45,000) improvements in sensitivity Kelstrup et al, in review 2017 over its Q Exactive HF MS Kelstrup et al, in review 2017. counterpart. To achieve the fastest possible fragmentation scans, the as a standard sample, loaded on column, and found that the team used a similar approach as the one used for optimizing Q Exactive HF-X MS delivered a 50% gain in the number of the Q Exactive HF MS (which consisted of increasing the peptides identified if the gradient length was 7.5 min. With injection time while monitoring the scan time between HCD much longer gradients (30 min), the gain drops to below 20%, spectra) to find the sweet spot for the parallel mode of opera- but that still translates to close to 4,000 unique proteins in half tion. The trade-off between resolution and speed had to take the time that it would have taken with the previous instrument. into account that signal-to-noise level (S/N) is sacrificed at the Furthermore, when using a 10X lower peptide load (only 100 lower resolution values. As a result, the fragment mass errors ng of the HeLa digest) at a resolution of 15,500, the number increased from about 3 ppm at a resolution of 30,000 to about of identifications drops by only 10% and is still above 18,000 7 ppm at a resolution of 7,500. unique peptides. Nevertheless, the team found that the majority of the frag- ment peaks were recorded with acceptable S/N ratios at a Application to Shotgun Proteomics resolution of 7,500, and that the best injection time was 11 The shotgun strategy involves an offline high-pH reverse-phase ms. The acquisition of HCD spectra under these conditions chromatographic fractionation of the samples to be analyzed can be done at a maximum rate of 41 Hz. At a resolution of and it identifies very large numbers of proteins in a relatively 15,000, the best injection time was 22 ms and the acquisition short time. For example, using a HeLa tryptic digest, up to 46 rate was 28 Hz. Finally, at a resolution of 45,000, they could fractions are collected and then analyzed with 30-min gradi- increase the injection time to 86 ms, which resulted in an ents in the Q Exactive HF MS, combining six runs to achieve acquisition rate of 10 Hz. close to 12,000 unique protein coding genes or 13,600 protein The faster sequencing achievable with many new spectrom- isoforms (see Figure 2). Looking at all protein isoforms and eters poses a challenge for the software that is used to detect peptide sequences identified, the researchers are confident peaks. This would be a limiting factor for reaching the expected that they are close to covering the expressed HeLa proteome full top MS/MS scan cycles. However, a new advanced peak in a comprehensive manner. detection (APD) algorithm capable of recognizing many more The approach also works well in the case of human tissues, peptide precursors and charge states enables the Q Exactive where it delivers very good coverage of the peptidome and HF-X MS to achieve full acquisition speeds, and up to 80% proteome of all cells the team studied, identifying between charge state annotations in full scan mode, compared to only 11,000 and 14,000 proteins. They could also identify transla- 30% with the previous version. tional modifications without specific enrichment. In the end, To benchmark the sensitivity of the new instrument, the they identified more than 10,000 phosphorylation sites in HeLa research team compared the number of unique peptides iden- cells, as well as more than 7,000 N-terminal acetylation sites, tified per minute using the fastest scan mode, at a resolution expanding the known literature by a factor of two. Compared of 7,500, between the Q Exactive HF MS and Q Exactive HF-X to the theoretical number of tryptic peptides in the size range MS instruments. The group used 1 μg of HeLa tryptic digest of 12 to 15 amino acids, this deep fractionation and analysis BENCHMARKING THE Q EXACTIVE HF-X MS FOR SHOTGUN PROTEOMICS Essentially complete HeLa proteome of 12,200 protein-coding genes Figure 2: Essentially complete HeLa proteome of 12,200 protein-coding genes. Bekker-Jensen et al, Cell Systems 2017. Bekker-Jensen et al, Cell Systems 2017 strategy can identify up to 75% of them. All together, the proteomics. After tissue sample preparation, a phosphopep- database generated by these studies represents the largest tide enrichment step involving the use of immobilized metal number of proteins and peptides reported so far in the large- affinity chromatography (IMAC) with TiO2 and antibodies is scale proteomics literature. needed (see Figure 4). It is also important to determine the With the new Q Exactive HF-X MS, the research team was stoichiometry of the phosphorylation site, so the absolute able to halve the gradient times from 30 to 15 minutes, which change in phosphorylation between conditions can be mea- translated into a reduction of the overall LC-MS analysis time sured, as well as the relative change. Finally, the functional from slightly over a day to only half a day, while still being phosphorylation sites and the phosphosignaling networks able to identify over 140,000 peptides covering 9,400 unique need to be identified, which requires a very advanced bioin- protein-coding genes. formatic analysis. These improvements are also applicable to phosphopro- The new scanning methods available on the Q Exactive teomics, where Olsen and co-workers have developed the HF-X MS, where a transient time of 96 ms gives a resolu- workflows that are now routinely used around the world (see tion of 45,000, enable significant improvements in overall Figure 3). Instead of performing quantitation by Stable Isotope speed. At this resolution level, the isobaric TMT reporter Labelling of Amino Acids in Cell Culture (SILAC) and using ions can still be resolved acceptably. In contrast, with the Isobaric Tag for relative and Absolute Quantitation (iTRAQ) previous instrument, a transient time of 128 ms had to be followed by hydrophilic interaction chromatography (HILIC) used. The slight drop in spectral quality was due to the fractionation, they use the shotgun approach described shorter transient time, which results in lower Andromeda previously. Stimulating the cells with epidermal growth factor phosphopeptide scores, is not considered significant. To (EGF) for different lengths of time allows them to obtain the demonstrate the benefits of the Q Exactive HF-X MS, five cell kinetic profiles for the individual phosphopeptides and phos- lines were analyzed in duplicate using TMT10-plex labeling phorylation sites of interest. HCD is then used to read out the agent and TiO2 for the IMAC step. The results show that phosphopeptide sequence and to pinpoint the phosphoryla- the number of phosphopeptide sites identified goes up to tion site in the peptides.
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