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Benchmarking the Q Exactive HF-X MS for Shotgun Proteomics

An Executive Summary

Hardware and software improvements allow researchers to push the speed and sensitivity limits of protein identification using quadrupole mass spectrometry.

IntroductionShotgun proteomics is a powerful technique for the global analysis of proteins and their post-translational modifications. The technique involves the proteolytic digestion of complex proteins 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 AnalysesThree advances in the hardware of the Q Exact i ve HF-X mass spectrom-eter account for the repor ted improvements in speed and sens i t i v i t y (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.

Jesper V. Olsen Professor

Novo Nordisk Foundation Center for Protein Research

University of Copenhagen

BENCHMARKING THE Q EXACTIVE HF-X MS FOR SHOTGUN PROTEOMICS

To compare the sensitivity of the Q Exactive HF-X mass spectrometer to the previous-generation instrument (Thermo Scienti f ic™ 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 shows that the Q Exactive HF-X mass spectrometer achieves an intensity increase of about 70%, and on average, up to two-fold improvements in sensitiv ity over i ts Q Exactive HF MS counterpart.

To achieve the fastest possible fragmentation scans, the team used a similar approach as the one used for optimizing the Q Exactive HF MS (which consisted of increasing the injection time while monitoring the scan time between HCD spectra) to find the sweet spot for the parallel mode of opera-tion. The trade-off between resolution and speed had to take into account that signal-to-noise level (S/N) is sacrificed at the lower resolution values. As a result, the fragment mass errors increased from about 3 ppm at a resolution of 30,000 to about 7 ppm at a resolution of 7,500.

Nevertheless, the team found that the majority of the frag-ment peaks were recorded with acceptable S/N ratios at a resolution of 7,500, and that the best injection time was 11 ms. The acquisition of HCD spectra under these conditions can be done at a maximum rate of 41 Hz. At a resolution of 15,000, the best injection time was 22 ms and the acquisition rate was 28 Hz. Finally, at a resolution of 45,000, they could increase the injection time to 86 ms, which resulted in an acquisition rate of 10 Hz.

The faster sequencing achievable with many new spectrom-eters poses a challenge for the software that is used to detect peaks. This would be a limiting factor for reaching the expected full top MS/MS scan cycles. However, a new advanced peak detection (APD) algorithm capable of recognizing many more peptide precursors and charge states enables the Q Exactive HF-X MS to achieve full acquisition speeds, and up to 80% charge state annotations in full scan mode, compared to only 30% with the previous version.

To benchmark the sensitivity of the new instrument, the research team compared the number of unique peptides iden-tified per minute using the fastest scan mode, at a resolution of 7,500, between the Q Exactive HF MS and Q Exactive HF-X MS instruments. The group used 1 μg of HeLa tryptic digest

as a standard sample, loaded on column, and found that the Q Exactive HF-X MS delivered a 50% gain in the number of peptides identified if the gradient length was 7.5 min. With much longer gradients (30 min), the gain drops to below 20%, but that still translates to close to 4,000 unique proteins in half the time that it would have taken with the previous instrument. Furthermore, when using a 10X lower peptide load (only 100 ng of the HeLa digest) at a resolution of 15,500, the number of identifications drops by only 10% and is still above 18,000 unique peptides.

Application to Shotgun ProteomicsThe shotgun strategy involves an offline high-pH reverse-phase chromatographic fractionation of the samples to be analyzed and it identifies very large numbers of proteins in a relatively short time. For example, using a HeLa tryptic digest, up to 46 fractions are collected and then analyzed with 30-min gradi-ents in the Q Exactive HF MS, combining six runs to achieve close to 12,000 unique protein coding genes or 13,600 protein isoforms (see Figure 2). Looking at all protein isoforms and peptide sequences identified, the researchers are confident that they are close to covering the expressed HeLa proteome in a comprehensive manner.

The approach also works well in the case of human tissues, where it delivers very good coverage of the peptidome and proteome of all cells the team studied, identifying between 11,000 and 14,000 proteins. They could also identify transla-tional modifications without specific enrichment. In the end, they identified more than 10,000 phosphorylation sites in HeLa cells, as well as more than 7,000 N-terminal acetylation sites, expanding the known literature by a factor of two. Compared to the theoretical number of tryptic peptides in the size range of 12 to 15 amino acids, this deep fractionation and analysis

Q Exactive HF-X MS: novel features

Optimized ion movements to minimize ion-transfer times

New Orbitrap resolution settings (7500 & 45,000)

New source inlet ion funnel design

Kelstrup et al, in review 2017

Figure 1: Q Exactive HF-X MS: novel features.

Kelstrup et al, in review 2017.

BENCHMARKING THE Q EXACTIVE HF-X MS FOR SHOTGUN PROTEOMICS

strategy can identify up to 75% of them. All together, the database generated by these studies represents the largest number of proteins and peptides reported so far in the large-scale proteomics literature.

With the new Q Exactive HF-X MS, the research team was able to halve the gradient times from 30 to 15 minutes, which translated into a reduction of the overall LC-MS analysis time from slightly over a day to only half a day, while still being able to identify over 140,000 peptides covering 9,400 unique protein-coding genes.

These improvements are also applicable to phosphopro-teomics, where Olsen and co-workers have developed the workflows that are now routinely used around the world (see Figure 3). Instead of performing quantitation by Stable Isotope Labelling of Amino Acids in Cell Culture (SILAC) and using Isobaric Tag for relative and Absolute Quantitation (iTRAQ) followed by hydrophilic interaction chromatography (HILIC) fractionation, they use the shotgun approach described previously. Stimulating the cells with epidermal growth factor (EGF) for different lengths of time allows them to obtain the kinetic profiles for the individual phosphopeptides and phos-phorylation sites of interest. HCD is then used to read out the phosphopeptide sequence and to pinpoint the phosphoryla-tion site in the peptides.

Challenges of In-Vivo PhosphoproteomicsPerforming phosphoproteomic analyses presents addi-tional challenges to the workflows developed for traditional

proteomics. After tissue sample preparation, a phosphopep-tide enrichment step involving the use of immobilized metal affinity chromatography (IMAC) with TiO2 and antibodies is needed (see Figure 4). It is also important to determine the stoichiometry of the phosphorylation site, so the absolute change in phosphorylation between conditions can be mea-sured, as well as the relative change. Finally, the functional phosphorylation sites and the phosphosignaling networks need to be identified, which requires a very advanced bioin-formatic analysis.

The new scanning methods available on the Q Exactive HF-X MS, where a transient time of 96 ms gives a resolu-tion of 45,000, enable significant improvements in overall speed. At this resolution level, the isobaric TMT reporter ions can still be resolved acceptably. In contrast, with the previous instrument, a transient time of 128 ms had to be used. The slight drop in spectral quality was due to the shorter transient time, which results in lower Andromeda phosphopeptide scores, is not considered significant. To demonstrate the benefits of the Q Exactive HF-X MS, five cell lines were analyzed in duplicate using TMT10-plex labeling agent and TiO2 for the IMAC step. The results show that the number of phosphopeptide sites identified goes up to slightly over 16,000 with the new instrument, compared to about 12,000 with the previous one. The analysis took just over six hours. Not only that, but the new instrument was able to cover much more of the low abundance phospho-peptides present in the samples.

Bekker-Jensen et al, Cell Systems 2017

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.

BENCHMARKING THE Q EXACTIVE HF-X MS FOR SHOTGUN PROTEOMICS

As a fur ther test of the speeds achievable with the new instrument, the team analyzed a 200-µg sample of HeLa digest, enr iched with TiO2, using four dif ferent scanning settings. They concluded that the best performing method, in terms of balancing speed and sensitivity, is the one at 28 Hz, with a resolution of 15,000. With these settings, they can identify close to 400 unique peptides per minute (see Figure 5) in a very reproducible manner.

Data-Independent Acquisition StrategiesGiven the current interest in DIA, the team explored the utility

In-Vivo Phosphoproteomics Challenges

2) Phosphopeptide identification and site localization

3) Phosphopeptide quantitation

4) Phosphorylation site stoichiometry determination

5) Functional phosphorylation sites and phospho-signaling networks

1)  Tissue sample preparation

& Phosphopeptide enrichment

Figure 4: In-Vivo Phosphoproteomics challenges.

Figure 3: Quantitative Phosphoproteomics technology.

Olsen and Mann, Mol Cell Proteomics 2013.

Olsen et al, Cell 2006. Francavilla et al, Mol Cell 2013. Francavilla et al, Nat Struct Mol Biol 2016. Olsen et al, Sci Signal 2010.

BENCHMARKING THE Q EXACTIVE HF-X MS FOR SHOTGUN PROTEOMICS

>5,000 unique phosphopeptides in 15min on Q Exactive HF-X

DDA versus DIA 30min LC-MS/MS on Q Exactive HF-X MS

Figure 5: >5,000 unique phosphopeptides in 15min on Q Exactive HF-X MS.

Figure 6: DDA versus DIA – 30min LC-MS/MS on Q Exactive HF-X MS.

BENCHMARKING THE Q EXACTIVE HF-X MS FOR SHOTGUN PROTEOMICS

of the Q Exactive HF-X MS in this promising field, taking advantage of the large, high-quality peptide libraries they already developed using data-dependent analysis (DDA). They focused on m/z values ranging from 385 to 1015, with 79 Thomson DIA MS/MS Windows and using an injection time of 22 ms and a resolution of 15,000 to generate comparative data. In just half an hour, the researchers identified more than 55,000 peptides and close to 6,000 proteins, which represents a two-fold increase over what is achievable with DDA (see Figure 6). As far as intensity and protein abundance coverage, they could cover one more order of magnitude with DIA than with DDA.

ConclusionsThe hardware and software innovations implemented in the new instrument lead to simultaneous improvements in acquisition speed and sensitivity, almost doubling what was

achievable with the previous generation hardware. More than 1,000 unique peptides per minute can now be identified in a very reproducible manner. With the new resolution settings, phosphoproteomics can be carried out using TMT10-plex and also in single-shot methods, where more than 5,000 peptides can be identified in 15 minutes of analysis time. Finally, very deep single-shot proteomes can be analyzed using DIA strategies.

Reference1. 1. C.D. Kelstrup, D.B. Bekker-Jensen, T.N. Arrey, A. Hogrebe,

A. Harder, and J.V. Olsen, J. Proteome Res. Publication Date (Web): November 29, 2017, http://pubs.acs.org/doi/10.1021/acs.jproteome.7b00602.

For Research Use Only. Not for use in diagnostic procedures.

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This executive summary is based on material presented in a webcast that can be viewed on demand here.