Delving Deeper Into the Proteome With Novel Mass Spectrometry Methods

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The ability to identify and accurately quantify peptides and proteins, in order to understand their biological functions, is largely dependent upon advances in analytical technology such as mass spectrometry (MS). Reproducible, rapid detection of protein expression patterns and ultimately the identification and characterization of protein behavior under specific environmental, physiological, or disease conditions are driven by the sensitivity and speed of commercially available mass spectrometers. In particular, advances in the efficiency of ion transmission,  and the speed at which instruments can process ions and identify and quantify the signals derived from them, are propelling developments across numerous proteomics applications.

MS-based proteomics is particularly useful for the large-scale analysis of post-translational modifications (PTMs), the investigation of protein–protein interactions, and the identification of clinical biomarkers. Clinical proteomics, which contributes to the diagnosis and treatment of a range of diseases, is one area undergoing development in the MS field. The in-depth profiling of complex patient samples, such as tissues, blood, or plasma, requires high specificity, sensitivity, and throughput. Quadrupole time-of-flight (QTOF) mass spectrometers are capable of providing deep proteome coverage with high reproducibility and are particularly suitable for proteomics applications due to their fast acquisition rates. This article highlights the integration of QTOF MS with trapped ion mobility spectrometry (TIMS) and the application of parallel accumulation serial fragmentation (PASEF)—an acquisition method that simultaneously offers improvements in speed, sensitivity, and robustness for proteome analysis, giving scientists the potential to discover more low-level, biologically significant proteins and validate them in research.

Speeding up shotgun proteomics

Shotgun (also known as discovery) proteomics is the primary bottom-up technique, which uses data-dependent acquisition (DDA) for unbiased proteome coverage. In DDA mode, the mass spectrometer scans the entire mass range (usually 300–1650 m/z) to obtain a mass spectrum (MS1) every few seconds. Each peptide needs to be fragmented by tandem mass spectrometry (MS/MS, MS2) for identification. The most abundant peptide peaks of each MS1 spectrum are successively isolated for fragmentation by a quadrupole or linear ion trap.

When using DDA, the researcher does not need to know the identity of the expected proteins in advance. This, in addition to fast spectra acquisition and its ability to fragment as many peptides as possible, makes DDA well-suited for discovery studies. However, a disadvantage of the method is that it is dependent on the sequencing speed of the mass spectrometer and, therefore, only a percentage of eluting peptides can be fragmented. Developments in MS technology and the introduction of TIMS in combination with the PASEF method have significantly increased the sequencing speed of shotgun proteomics with DDA, facilitating large-scale protein identification and quantification and protein biomarker discovery.

PASEF is an emerging method that enables several precursor ions to be stored in parallel in a trapped ion mobility device, which can then be followed by serial fragmentation. It allows hundreds of MS/MS events per second, with improved sensitivity, providing a several-fold speed and sensitivity increase compared to traditional techniques¹ and increased DDA rates. As a result of implementing PASEF, 70% of the overall precursor population can be targeted and potentially identified with MS/MS.²

Handling shotgun proteomics data

Very large datasets are produced in MS-based proteomics, making interpretation of such data challenging. Analysis involves feature detection and processing, peptide identification, protein identification, and quantification. Freely available software, such as MaxQuant (Max-Planck Institute of Biochemistry, Martinsried, Germany), has been developed to overcome these challenges. A completely open data file format specific to the timsTOF Pro (Bruker Daltonics, Billerica, MA) allows researchers  to work directly with the raw data when using software for shotgun proteomics analysis. PEAKS Studio (Bioinformatics Solutions, Waterloo, Ontario, Canada) is another software package supporting MS data analysis, also capable of processing data from the timsTOF Pro, which has a novel de novo sequencing algorithm that is integrated into the traditional database searches to ensure a complete interpretation of raw spectral data. The solution handles the complexity of  PASEF data  to advance proteomics research through peptide/protein identification and quantification, peptide mapping, and PTM characterization.

PASEF in practice

The increase in sequencing speed and sensitivity, as well as the additional dimension of separation offered by applying the PASEF method to a TIMS-QTOF MS, has been shown to alleviate some of the challenges that arise from DDA shotgun proteomics. A group at the Max Planck Institute for Biochemistry demonstrated the use of PASEF on TIMS-QTOF MS for shotgun proteomics. Their most recent study reveals different precursor selection schemes for shotgun proteomics to optimally allocate over 100 fragmentation events per second.¹ The group also highlighted the high sensitivity achievable with minimal sample volumes.

Complex human cervical cancer cell (HeLa) peptide mixtures resulting from a LysC and trypsin digest were separated using LC and analyzed with the timsTOF Pro. An algorithm was developed with the aim to maximize the number of precursors per acquisition cycle that can be successfully identified. Figure 1 shows three dimensions: precursor m/z, signal intensity, and ion mobility for real-time PASEF precursor selection. From this 100-msec TIMS scan, the algorithm selected 50 unique precursor ions for fragmentation in the subsequent PASEF scans.

Further experiments to test the speed and sensitivity of the instrument at low sample volumes showed that over 800,000 fragmentation spectra are easily achievable in standard 120-min LC runs. Using the PASEF method, more than 2900 proteins could be identified in 30-min runs of only 10 ng HeLa digest, showing its high sensitivity (Figure 2).¹

Figure 2 – Rapid and sensitive HeLa proteome measurements. a) Total ion chromatograms of the 60-min gradient and three different sample amounts on column. b) Average number of protein groups identified and quantified with a CV <20% in 60-min single runs (N = 3). c) Total ion chromatograms of the 30-min gradient and three different sample amounts on column. d) Average number of protein groups identified and quantified with a CV <20% in 30-min single runs (N = 3). e) Total  ion chromatogram of a 5.6-min gradient with 50-ng HeLa digest on column. f) Number of protein groups identified in 10 replicate injections with the 5.6-min gradient.

The high sequencing rate consistently delivered at over 100 Hz by the timsTOF Pro would usually lead to short ion collection times and, therefore, poor spectrum quality. However, with the PASEF method, the ions are focused in time and space by the trapped ion mobility separation (TIMS), and the full scan speed of TOF instruments can be leveraged while maintaining very high sensitivity, enabling the identification of thousands of protein groups in single runs from human cancer cell lines with minimal sample volumes and high quantitative accuracy.

MS-based clinical proteomics

In clinical applications, many types of patient samples are only available in very limited quantities, presenting a challenge to most protein identification technologies. The higher speed and sensitivity shotgun proteomics enabled by the PASEF technology make it particularly suited for analysis where very limited sample is available. The increase in sensitivity afforded by PASEF with TIMS-QTOF MS can drive biomarker discovery, as protein biomarker candidates may be present at the lower end of the plasma protein concentration range.²

Clinical proteomics in the future

MS-based proteomics has provided important insights into the composition, regulation, and function of molecular pathways for a deeper understanding of complex biological processes. Technological developments such as the PASEF method for TIMS-QTOF MS protein analysis will continue the positive impact of MS-based proteomics across biology and medicine by virtue of its precise identification and quantification of thousands of proteins from complex samples.

Personalized medicine is a rapidly expanding field, and the use of MS- based proteomics is expected to contribute to its advance, as not all disease states have genetic markers. Such uses of MS for clinical proteomics could therefore advance the application of targeted therapy, and the measurement of disease-relevant biomarkers could also be further developed in the future with MS-based proteomics, especially using TIMS-QTOF MS with PASEF.

References

1. Meier, F.; Brunner, A.-D. et al. Online parallel accumulation—serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer, 2018; https://doi.org/10.1101/336743.
2. Meier, F.; Beck, S. et al. Parallel accumulation−serial fragmentation (PASEF): multiplying sequencing speed and sensitivity by synchronized scans in a trapped ion mobility device. J. Proteome Res. 2015, 14, 5378–87.

Gary Kruppa is vice president of Proteomics, Bruker Daltonics Inc., 40 Manning Rd., Manning Park, Billerica, MA 01821, U.S.A.; tel.: 978-663-3660; e-mail: [email protected]; www.bruker.com. Scarlet Koch is global market manager, Proteomics, Bruker Daltonik GmbH, Bremen, Germany.