The era of 4D-Omics
Unlocking the value of the 4th dimension
Mass spectrometry (MS)-based proteomics is the method of choice for the identification and quantification of thousands of proteins. However, the complete coverage of proteomes remains challenging due to the limited speed, sensitivity and resolution of current mass spectrometers.
The timsTOF Pro 2 uses the parallel accumulation serial fragmentation (PASEF®) acquisition method to provide extremely high speed and sensitivity, requiring only minimal sample amounts to reach new depths in proteomics.
Trapped ion mobility spectrometry (TIMS) is first and foremost a separation technique in the gas phase. This resolves sample complexity through an added dimension of separation in addition to high performance liquid chromatography (HPLC) and mass spectrometry, increasing peak capacity and confidence in compound characterization.
Equally important, the TIMS device also accumulates and concentrates ions of a given mass and mobility, enabling a unique increase in sensitivity and speed.
A near 100% duty cycle can be achieved with the dual-TIMS technology facilitating accumulation in the front section, while ions in the rear section are sequentially released depending on their mobility. This process of parallel accumulation serial fragmentation (PASEF®) enables collisional cross section (CCS) analysis.
CCS-enabled analysis opens up many further analytical possibilities, from greater certainty of compound identification to confident library matching and lower false discovery rates (FDRs) in large datasets.
Introducing the timsTOF Pro 2 mass spectrometer with our newest generation TIMS analyzer and a completely reworked stainless steel stacked ring ion guide (SRIG). The new design offers 3 times higher ion capacity. A new ion cooling multipole for ion pre-conditioning and improved ion injection into the quadrupole together with further simplified ion optics for more efficient ion transfer from the TIMS cell to the TOF analyzer, further maximize ion transfer and sensitivity both in MS and PASEF MS/MS.
The unique design of the TIMS cell means that ions are released from the second section of the TIMS analyzer depending on their mobility, while the further incoming ions can be accumulated in parallel in the first part of the TIMS analyzer.
This parallel accumulation technology achieves a duty cycle of nearly 100%, resulting in nearly no ion loss.
Redesigned MS/MS technology to meet the speed requirements of proteomics. Peptide ions are separated using trapped ion mobility spectrometry, eluted (~ 100 ms) and detected in the quadrupole time of flight (QTOF), generating the TIMS MS heat map. In the parallel accumulation serial fragmentation (PASEF®) method the same TIMS separation is used: the quadrupole isolates a certain ion species during its elution and immediately shifts to the next precursor. Parent and fragment spectra are aligned by mobility values.
PASEF® technology can achieve a sequencing speed of >100 Hz and the MS/MS spectra quality of the low abundant peptides can be increased by selecting them several times.
PASEF®: the perfect fit for shotgun proteomics
The timsTOF Pro 2 powered by PASEF® offers a sequencing speed of >100 Hz without losing sensitivity or resolution. This is achieved by synchronizing the quadrupole isolation mass window with the elution time of the specific peptide packages from the TIMS funnel.
No cleaning required
Many MS instruments used for proteomics applications require monthly cleaning when run 24 hours a day on large sample cohorts. Performance degradation is
noticeable even over shorter time periods. The superior robustness of the timsTOF Pro 2 means that the instrument can be run 24/7 over many weeks without noticeable loss of sensitivity or other performance metrics.
"Since we started to work with the TimsTOF Pro 26 months ago in February 2019, we have run more than 25000 LCMS samples, of which about 5000 have been non-depleted plasma digests. We had virtually zero downtime so far."
PaSER (Parallel Search Engine in Real-time) is a combined hardware and software solution enabling fully integrated real-time database searches and results based sample queue management.
PaSER delivers results with uncompromising speed, including PTM searches. By utilizing GPU based searches, PaSER delivers the same results in real-time or offline mode without the need to utilize simplified algorithms or intermediates. This uncompromised search speed of PaSER allows you to have results on hand, seconds after the acquisition ends, Run & Done! PaSER effectively removes the data analysis bottleneck introduced by large sample cohorts and greater throughput.
Additionally, real-time LFQ quantification can also be performed across PaSER acquired data sets making the transition into quantitative proteomics instantaneous. Visualization of mobility offset mass aligned (MOMA) features using TIMS Viz allows the user to immediately identify and characterize isobaric and near-isobaric peptides that are only visible and identifiable by 4D-Omics.
dia-PASEF is both more sensitive and selective than traditional DIA approaches as it applies the PASEF principle to data independent acquisition, combining the advantages of DIA with the inherent ion efficiency of PASEF.
TIMS separation increases selectivity, excludes singly charged precursors from fragmentation and cleans up the sample by concentrating signals from noise.
Making use of the correlation of molecular weight and CCS coded information from the dual-TIMS funnel, dia-PASEF enables most confident compound identification. Over the entire LC-MS/MS dia-PASEF runs a perfect data cuboid is created containing m/z, ion mobility (CCS), retention time and intensity.
With the robust SRIG (Stacked Ring Ion Guide) configuration and new optimized standard dda-PASEF methods timsTOF Pro 2 provides unprecedented depth of proteome coverage in single shot proteomics. From inhouse HEK tryptic digests of 200 ng in 60 minute gradients using an Aurora-25 cm columns, more than 7000 protein groups and 60,000 peptides were identified.
timsTOF Pro 2 thus provides in-depth proteome coverages for everyday cell line proteome quantification experiments directly by database searching and matching between the runs without the need for any spectral library. Different database search strategies resulted in very comparable results. PaSER, enabling real time protein identifications, and MaxQuant resulted in similar ID numbers on both protein and peptide level.
In comparison with standard selected and parallel reaction monitoring (SRM and PRM), prm-PASEF increases the number of peptides that can be targeted in a single acquisition method, without compromising the selectivity or the sensitivity.
Targeted mass spectrometry (MS) is a powerful technique that is used in proteomics experiments – to verify biomarker candidates in large sample cohorts, for example. This increases sensitivity compared to data dependent acquisition (DDA) and data independent acquisition (DIA). This technique is limited by a necessary compromise between the number of targets measured in a single run, the duration of the liquid chromatography separation stage and the overall sensitivity. It is only possible to achieve complete data for a large number of targeted peptides either by running longer chromatography separation or compromising on MS sensitivity and selectivity.
prm-PASEF increases the number of peptides that can be targeted in a single acquisition by benefiting from the 4th dimension of separation using Bruker’s timsTOF Pro 2 to improve selectivity and sensitivity, adding the speed of PASEF to increase the number of precursor targets.
Proteome quantification using low sample amounts is crucial for a growing number of biological applications such as specialized cells, rare cell populations, or fine needle aspiration tumor biopsies. Proteome quantification of such low sample amounts using a sensitive mass spectrometer is crucial. From 20 ng of HeLa (Pierce) peptides measured on Aurora-25 cm column in 30 minute gradients, PaSER – realtime search engine – identifies more than 4200 protein groups and close to 30,000 peptides.
Data independent acquisition using standard dia-PASEF methods provides reproducible identifications in multiple runs. Three different dia-PASEF window schemes allow for the quantification of close to 8000 protein groups and more than 70,000 peptide sequences in 60 minute gradients using Aurora-25cm columns, while demonstrating quantitative precision.
CCS-enabled quantification of proximal phosphorylation sites
The high sensitivity, sequencing speed and reproducibility of dia-PASEF on the timsTOF Pro 2 even enables quantitative phosphoproteomic analyses of limited sample amounts. Label-free quantification of phosphoproteomes is feasible from as little as 25 μg of total protein obtained from mouse brain samples. dia-PASEF analysis of enriched phosphopeptides using a 30 SPD (samples per day) Evosep method resulted in the identification of up to 4473 unique phosphopeptides across three enrichment replicates. These results hold further promise for the application in needle biopsies, complementing cancer proteogenomics data with information on signal transduction. Results are provided courtesy of Prof. Stefan Tenzer.
Analyze cell signaling where sample amounts are limited
At the point of chromatographic co-elution, quantification of phospho-peptide (p-peptide) isomers is not possible in traditional proteomics approaches without CCS information due to the isobaric nature and signal overlay. PASEF analyses from standard 150 μg TiO2-based enrichment workflows identifies 27,768 phosphopeptides, as shown on the right and reveals the benefits of ion mobility separation with Mobility Offset Mass Aligned (MOMA). From 1946 identified co-eluting isomers, 20% could be fully separated by TIMS, enabling a better understanding of proximal protein phosphorylation sites.
"Besides its high sensitivity, a unique aspect of the timsTOF Pro instrument is its capability to resolve positional phosphorylation isomers in the gas phase, thus providing more detailed insight into signaling pathways."
For Research Use Only. Not for use in clinical diagnostic procedures.