Software innovations in four-dimensional mass spectrometric data analysis
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VOL. 32 NO. 6 (2020)
ARTICLE
Software innovations in
four-dimensional mass
spectrometric data analysis
Jürgen Coxa and Gary Kruppab
a
Research Group Lead of Computational Systems Biochemistry, Max Planck Institute of Biochemistry, Am
Klopferspitz 18, 82152 Martinsried, Germany. cox@biochem.mpg.de; https://orcid.org/0000-0001-8597-205X
b
Vice-President, Proteomics, Bruker Daltonics
Over the last two decades, significant MaxQuant possesses a large ecosys- widespread adoption of Bruker’s timsTOF
advances in technology and new meth- tem of algorithms for comprehensive Pro instrument, which was designed
odologies have made proteomics an data analysis. It incorporates the peptide to deliver greater sensitivity, selectiv-
extremely powerful tool for protein scien- search engine Andromeda and, coupled ity and MS/MS acquisition speeds for
tists, biologists, and clinical researchers.1 with Perseus, was developed to offer a proteomics research. The novel design
As analytical instrumentation continues complete solution for downstream bioin- allows for ions to be accumulated in
to evolve, more data is produced with formatics analysis.2 MaxQuant performs the front section, while ions in the
each technical advancement in proteom- quantification with labels and via the rear section are sequentially released
ics research. That, of course, also creates MaxLFQ algorithm on label-free data, and depending on their ion mobility, and in
new challenges for bioinformatics soft- achieves high peptide mass accuracies subsequent scans selected precursors
ware development. thanks to its advanced non-linear recali- can be targeted for MS/MS. This process
The modern high-throughput mass bration algorithms. is called Parallel Accumulation Serial
spectrometry (MS) -based proteom- Fragmentation, or PASEF®.3
ics methods that are required to gain MaxQuant for four- The unique trapped ion mobil-
deeper insights into biological processes dimensional proteomics ity spectrometry (TIMS) design allows
produce enormous amounts of data. This MaxQuant is often used for liquid chro- researchers to reproducibly measure the
raw data necessitates powerful, auto- matography coupled with tandem mass collisional cross-section (CCS) values for
mated computer-based methods that spectrometry (LC-MS/MS) shotgun all detected ions, and those can be used
provide reliable identification and quan- proteomics—a method of identifying to further increase the system’s selec-
tification of proteins. proteins in complex mixtures to provide tivity, enabling more and more reliable
a wider dynamic range and coverage of relative quantitation information from
Quantitative proteomics proteins. complex samples and short gradient
software Shotgun (or bottom-up) proteomics analyses.
Freely available for academic and non- is the most commonly used MS-based The addition of TIMS to LC-MS based
academic researchers, many labora- approach to study proteins by digesting shotgun proteomics, using PASEF,
tories across the globe benefit from them into peptides prior to MS analysis. has the potential to boost proteome
the MaxQuant quantitative proteom- Ion mobility can add a further dimen- coverage, quantification accuracy and
ics software package’s precise protein sion to LC-MS based shotgun proteomics dynamic range, resulting in fast ultra-
and peptide quantification algorithms. that has the potential to boost proteome sensitive analysis. The increase in
The software was developed by the coverage, quantification accuracy and speed resulting from PASEF technology
Computational Systems Biochemistry dynamic range. However, this additional allows more samples to be analysed in
group at the Max Planck Institute of information requires suitable software a shorter time frame, but also generates
Biochemistry (MPIB) in Martinsried, that extracts the information contained vast amounts of spectral data, creating
Munich, Germany. The group also devel- in the four-dimensional (4D) data space challenges when dealing with large
oped Perseus, a software platform that spanned by m/z, retention time, ion sample cohorts.
supports researchers in the interpretation mobility and signal intensity. MPIB software developers adapted
of protein quantification and interaction The impact of the addition of the ion the MaxQuant shotgun proteomics
data as well as data on post-translational mobility dimension on the proteomics workflow to extract this abundance
modifications (PTMs). field can be seen, for example, in the of information from the timsTOF Pro
14 SPECTROSCOPYEUROPE www.spectroscopyeurope.com
VOL. 32 NO. 6 (2020)
ARTICLE
Figure 1. Screenshot of MaxQuant quantitative proteomics software package designed for analysing large mass spectrometric data sets. Source:
Maxquant.org
data, making it possible to manage 4D Emerging applications for research proteomics will be one of the
features in the space spanned by reten- 4D-Proteomics main applications of 4D-Proteomics
tion time, ion mobility, mass and signal Powerful developments in MS tech- in the future, and they are working
intensity that benefit the identification nology have led to the expansion of with several clinical groups to bring
and quantification of peptides, proteins MaxQuant and the software’s ability to MS-based proteomics into clinical prac-
and PTMs. meet future needs in the proteomics tice. However, the analysis of proteom-
TIMS is challenging for software devel- field. These improvements are helping ics data from samples derived from
opers because it is not just one piece of researchers develop new capabilities and patients requires special computational
new information—it adds an additional applications by delivering more sensitivity strategies. The problems that need to be
dimension. The updated MaxQuant and selectivity for the identification and addressed include: how to extract mean-
4D-Proteomics workflows can process quantification of peptides, proteins and ingful protein expression signatures from
data produced via PASEF, data-indepen- PTMs. Instrumentation advances have data with high individual variability, how
dent acquisition (dia)-PASEF and Mobility also bolstered the ongoing development to integrate the genomic background of
Offset Mass Aligned (MOMA). of MaxQuant. The MPIB software devel- the patients into the analysis of proteom-
Adding another dimension can opment team’s goal is always to expand ics data, and how to determine biomark-
lengthen algorithm processing times, and improve MaxQuant to meet the ers and properly estimate their predictive
creating a significant challenge for soft- complexity of biological processes and power.
ware development with 4D-Proteomics. novel MS instruments. To answer these questions, the MPIB
As a result, the team optimised computa- software developers are working to make
tion time in MaxQuant to overcome this, Clinical research proteomics use of machine learning algorithms to
so users can achieve good results in a The MPIB Computational Systems classify patients and employ feature
reasonable time frame. Biochemistry team believes clinical selection algorithms to extract predictive
www.spectroscopyeurope.com SPECTROSCOPYEUROPE 15
VOL. 32 NO. 6 (2020)
ARTICLE
Figure 2. Design of the timsTOF Pro instrument. Source: www.Bruker.com
protein signatures. The extra clinical test
engine provides a challenge for the soft-
ware. It is not clear yet if proteomics will
be a guide to which molecules to look at,
or if it will be a major component of clini-
cal diagnostics.
Single-cell proteomics
While today’s laboratories generate large
data sets from single-cell genomic and
single-cell transcriptomic research, single
cell proteomics (sc-proteomics) is a
nascent field. It holds the potential to
enable researchers to detect and quanti-
tate the proteins in single cells, avoiding
the need to infer proteins from cellular
messenger-RNA levels.4 That also creates
new challenges for computational analy-
sis.
MPIB researchers are looking ahead
to future needs as sc-proteomics devel-
ops, closely examining emerging tech-
nologies to establish quantification
standards. The software developers
believe sc-proteomics will be achiev-
able in the next few years. The two main
advances enabling this will be improved
sensitivity in the instrumentation and the
software to work with it.
Figure 3. Example showing how dia-PASEF can efficiently fragment nearly all peptide ions
Data-independent eluting at a given retention time. Figures courtesy of: F. Meier, A.-D. Brumer, M. Frank, A. Ha, I.
acquisition Bludau, E. Voytik, S. Kaspar-Schoenefeld, M. Lubeck, O. Raether, R. Aebersold, B. Collins, H.-L. Rost
Recent advances in data-independent and M. Mann, “Parallel accumulation – serial fragmentation combined with data-independent
acquisition (DIA) sensitivity have encour- acquisition (diaPASEF): Bottom-up proteomics with near optimal usage”, bioRxiv 656207 (2020).
aged the MPIB Computational Systems https://doi.org/10.1101/656207
16 SPECTROSCOPYEUROPE www.spectroscopyeurope.com
VOL. 32 NO. 6 (2020)
ARTICLE
Biochemistry team to integrate DIA work- of proteomics, which could extend the References
flows into MaxQuant using machine feasibility of applying 4D-Proteomics 1. J. Cox and M. Mann, “Quantitative,
learning algorithms. The success of DIA for clinical research applications, as high-resolution proteomics for data-
relies on key instrumental capabilities— mentioned previously. driven systems biology”, Ann. Rev.
namely resolution, sensitivity, accuracy Biochem. 80, 273–299 (2011).
and dynamic range uncompromised by Conclusion https://doi.org/10.1146/annurev-
a fast-spectral acquisition rate. Bioinformatics software development biochem-061308-093216
DIA has been implemented in the is a constantly changing field that will 2. J. Cox, N. Neuhauser, A. Michalski,
timsTOF Pro in a way that takes advan- see more technological advancement R.A. Scheltema, J.V. Olsen and M.
tage of the speed and sensitivity of in the future. As analytical instrumenta- Mann, “Andromeda: a peptide search
TIMS and PASEF, in a method called tion advances, the MPIB Computational engine integrated into the MaxQuant
dia-PASEF®. The 4D nature of the dia- Systems Biochemistry team must deal environment”, J. Proteome Res.
PASEF data is an advantage for DIA soft- with even larger amounts of informa- 10(4), 1794–1805 (2011). https://
ware developers, who can make use of tion on the software side because these doi.org/10.1021/pr101065j
the additional ion mobility dimension instruments continue to expand their 3. F. Meier, A.D. Brunner, S. Koch, H.
for alignment and extraction of features. dynamic range and capabilities. Koch, M. Lubeck, M. Krause, N.
Such recent technological advances, These improvements to the MaxQuant Goedecke, J. Decker, T. Kosinski,
as well as developments in DIA meth- software platform are possible due M.A. Park, N. Bache, O. Hoerning,
ods, have provided new opportunities to important collaborations between J. Cox, O. Räther and M. Mann,
for the MPIB Computational Systems the MPIB Computational Systems “Online Parallel Accumulation-Serial
Biochemistry research group. Biochemistry research team and other Fragmentation (PASEF) with a novel
The MaxQuant developers are enthu- institutions and industry leaders. These trapped ion mobility mass spectrom-
siastic about the platform’s upcom- collaborations include projects designed eter”, Mol. Cell. Proteomics 17(12),
ing DIA capability. DDA and DIA are to improve proteomics technologies, with 2534–2545 (2018). https://doi.
becoming comparable because of MaxQuant providing the computational org/10.1074/mcp.TIR118.000900
improved sensitivity in instrumentation. tools necessary to analyse data acquired 4. V. Marx, “A dream of single-cell prot-
The hardware is becoming simpler for on new and emerging hardware plat- eomics”, Nat. Methods 16, 809–812
users, but data has been much more forms. The results of these collaborations (2019). https://doi.org/10.1038/
challenging because the sof t ware will be used to develop future versions of s41592-019-0540-6
must find which fragments belong to the software to optimise 4D-Proteomics
which molecule. Addressing these chal- workflows.
lenges will provide a deeper coverage
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