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Targeted Proteomics with the diaPASEF Workflow

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Bruker has announced the release of the diaPASEF workflow, a novel data-independent acquisition (DIA) method on the timsTOF Pro platform. The timsTOF Pro leverages trapped ion mobility spectrometry (TIMS) and ‘parallel accumulation - serial fragmentation’ (PASEF), developed in an intensive collaboration over several years with the group of Professor Matthias Mann at the Max Planck Institute.

The new diaPASEF workflow uses overlapping windows in the ion mobility domain to trigger MS/MS, efficiently using the quadrupole to transmit precursor ions at high sensitivity. Using the inherent duty-cycle advantage of PASEF, diaPASEF typically results in a further 30% improvement in protein identifications, now with over 7,500 proteins identified in a 120 minute single-shot experiment on 200 ng of HeLa digest. diaPASEF also alleviates the so-called ‘missing value problem’ in stochastic, data-dependent acquisition (DDA) methods, and thereby improves data completeness and sensitivity.

In addition to the much more efficient ion usage in diaPASEF, when compared to traditional DIA methods, the timsTOF Pro provides the additional dimension of ion mobility, or collision cross sections (CCS), which all CCS-aware proteomics software solutions now take advantage of. Besides retention time alignment, the timsTOF Pro also allows simultaneous ion mobility alignment of precursors and fragments in diaPASEF, improving the reliability and specificity of the assignments even further.

Further results on the quantitative performance of diaPASEF will be presented at HUPO 2019: Using label-free quantification (LFQ) on a mixture of three proteomes (HeLa, Yeast, E.coli) and Spectronaut software, more than 8,000 proteins could be reliably identified and quantified with at least two peptides at a false discovery rate of 1%.

Dr. Roman Fischer, Principal Investigator at the Nuffield Department of Medicine, Target Discovery Institute at University of Oxford, stated: ”The arrival of the timsTOF Pro with PASEF has completely changed the game for clinical proteomics. Suddenly we can scale up clinical studies by a factor of 10x or more, and even improve on robustness, data depth and completeness. We are excited about the prospects for diaPASEF with short gradients for our clinical research.”

Work-in-Progress Novel 3D-targeted PRM workflow with additional ion mobility targeting

For targeted quantitative proteomics, Bruker has also introduced a novel 3D-targeted Parallel Reaction Monitoring (PRM) method on the timsTOF Pro as work-in-progress at HUPO 2019. In traditional 2D PRM experiments, retention times and masses are used to target and quantify lists of targeted protein biomarkers. A problem in 2D PRM is that interfering peptides that co-elute with similar precursor masses can be co-fragmented, which can reduce quantitative performance, especially near the lower limits of quantitation in complex matrices, like plasma.

The timsTOF Pro with its 4D proteomics capabilities now can target proteins in three dimensions, namely retention time and mass, plus by universal, molecule-specific and precise CCS values. This additional 3D-targeting for PRM in ion mobility, combined with the sensitivity enhancement provided by TIMS/PASEF, and the >100 Hz MS/MS speed at 50,000 mass resolution of the timsTOF Pro, means that more proteins can be ‘3D-targeted’ by PRM with sensitivity and quantitation.

Dr. Gary Kruppa, Bruker’s Vice President of Proteomics, said: “With diaPASEF we expect accelerated development of new applications and more exciting results. The development of fast and user-friendly software for library generation and results analysis also is key to the adoption of diaPASEF. We are very pleased to have such widespread support for diaPASEF software from our collaboration partners. Combined with new developments in 3D-targeted PRM for proteomics, the timsTOF Pro covers ever more CCS-aware 4D proteomics workflows in a unique way.ˮ

Bruker has begun collaborating with early adopters on 3D-targeted PRM quantitation for proteomics with new beta-software to evaluate performance and obtain user input.

Dr. Jarrod Marto, Associate Professor at the Dana-Farber Cancer Institute, Harvard Medical Center, and Brigham and Women’s Hospital stated: “We are excited to be working with Bruker on combining first-in-class acquisition speed with ion mobility to drive new 3D-targeted PRM workflows on the timsTOF Pro. Our initial results are very promising, and we are very pleased with the collaborative effort to push PRM to new levels of performance.”