With the introduction of the timsTOF platform the next evolution in technology is achieved by adding an additional dimension of gas phase ion separation via the use of TIMS coupled to UHR-QTOF technology. This results in the transition from 3D-Proteomics (retention time, m/z and MS/MS fingerprint) into 4D-Proteomics™ (addition of intrinsic CCS values).

4D-MBR and dia-PASEF

4D-MBR and dia-PASEF®: Ushering in the area of 4D-Proteomics™

The addition of the ion mobility dimension in the timsTOF Pro and timsTOF fleX delivers more benefits than an increase of sensitivity, selectivity and MS/MS acquisition speed. The unique tims design allows to reproducibly measure the Collisional Cross Section (CCS) values for all detected ions, and those can be used to further increase the system’s selectivity, enabling more and more reliable relative quantitation information from complex samples and short gradient analyses.

4D-MBR: resolving the missing value problem

4D-MBR: resolving the missing value problem for short gradients LFQ experiments

DIA is the most widely used acquisition strategy to resolve the missing value problem. However, using it requires the construction of spectral libraries – which can be timely and difficult when it concerns an uncommon organism – and traditional DIA methods are too slow to be compatible with short gradient analysis. In such cases, the PASEF-LFQ delivers speed, flexibility and reliability. Nonetheless, even with > 100Hz MS/MS, some peptides might not be selected for fragmentation in every analysis, especially while using short gradients.

The Match Between Runs (MBR) approach has been developed to resolve this missing value issue. MBR extracts intensity information for all peptides to be quantified using narrow m/z and retention time windows. This combination of filters, previously suggested as not being specific enough, is resulting in inaccurate ratio measurements. 4D-MBR adds an extra CCS filter to the first m/z and retention time ones, allowing for much greater specificity as illustrated in our application note LC-MS 151.

4D-MBR applied on high-throughtput plasma proteomics Outcome for the quantitation of 192 patient and 20 QC plasma samples digests, separated with an 11.5 min gradient using an Evosep One LC system. (click to enlarge)


dia-PASEF®: taking DIA analysis to the next level

The combination of DIA with PASEF® allows to compensate for the traditional DIA pitfalls: by using a pattern of m/z isolation windows within consecutive tims events, the percentage of ions used in the dia-PASEF® can be greatly increased (reaching up to 100% for low complexity samples, and still 5X higher than with traditional DIA methods using comparable isolation windows size and m:z range for high complexity samples). The dia-PASEF® cycle time can be reduced to make it compatible with short gradient separation while preserving a high selectivity. It benefits from the tims space concentration effect that allows for increased sensitivity and takes DIA selectivity to the next level with full 4D-Proteomics™ support.

dia-PASEF® data can now be processed by using OpenSwath & MOBI-DIK or Biognosys’s Spectronaut software. Learn more by reading our application notes LC-MS 157 and LC-MS 160.