Juan Rojas Juan Rojas was introduced to his mass spectrometry journey while working at INDICASAT AIP (Panama) as a mass spectrometry technician assisting in diverse natural products discovery, plant metabolites profiling, and species identification projects. Later on, he obtained a MSc degree at University of Warwick (United Kingdom) with a special focus on analysis of big data and mass spectrometry. Since 2018, he has continued to develop these research interests at Universität Leipzig as a Ph.D. candidate under the tutelage of Prof. Dr. Ralf Hoffmann. The focus of Juan’s work is on the use LC IMS MS/MS to develop new methods that allow identifying low abundance carbonylation PTMs in human plasma and quantifying peptides carrying these PTMs. Due to all the data quality validation features that Skyline offers, crucial for the analysis of complex LC-IMS-MS/MS data, Juan has become an experienced and outspoken user of the platform’s transparent and flexible data analysis capabilities.

Skyline for the parallel analysis of LC-TWIMS-MS/MS DDA and DIA Data

Electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) allows rapid analyses of complex mixtures, for example in bottom-up proteomics, with high resolution and high mass accuracy, especially when coupled on-line to an HPLC. The sensitivity for these TOF-instruments depends on the transfer rate of ions, which are continuously generated in the ESI source, into the TOF mass analyzer by orthogonal acceleration (oa). The efficiency of this duty cycle depends upon the m/z-range to be scanned and is limited by the highest m/z-value to be recorded, as the corresponding ions travel with the lowest speed. However, when a travelling wave ion mobility spectrometer (TWIMS) is coupled to the TOF, the drift time of each ion can be synchronized to the pusher and the duty cycle of the TOF. Thus, fragment ions generated after collision induced dissociation (CID) can be separated in the TWIMS cell before transferring them to the TOF, for example on a Q-TWIMS-TOF SYNAPT G2-Si mass spectrometer (Waters Corp., Manchester, UK). This instrument allows using the drift time of each segment to optimize the parameters of the following ion transfer and detection period. This improves the sensitivity for peptide fragment ions acquired in a data dependent acquisition (DDA) mode up to tenfold. However, DDA modes are limited by their stochastic nature, which can be overcome by using data independent acquisition (DIA) modes, such as MSE, which again is limited in sensitivity due to the orthogonal acceleration in front of the TOF region. Thus, we developed a novel acquisition mode by combining TWIMS-MS/MS, which is currently used only in combination with DDA, with MSE. This newly designed signal enhancement MSE (seMSE) was evaluated in comparison to five other DDA and MSE data acquisition techniques by analyzing two complex protein digests and considering the number of identified peptides and the corresponding peak areas. The six data sets of each sample were processed in parallel using Skyline, as it was the only software package capable to process the different data formats and compare the quality of the generated peptide spectral libraries and the signals recorded by each MS method. Among the tested MSE methods, seMSE provided the best fragment ion sensitivity, but faced similar limitations as non-IMS MSE methods, i.e., a limited spectral library of confidently assigned peptides. Nevertheless, this limitation was overcome by Skyline using the spectral libraries generated with other MS methods, especially those generated with DDA and fragment ion TWIMS. Thus, seMSE provided around tenfold higher signal intensities allowing a more reliable quantitation together with improved lower limits of quantitation without reducing the number of signals to be quantitated.