MOTIVATION: The data independent acquisition (DIA) mass spectrometry (MS) method is increasingly popular in the field of proteomics. But the loss of the correspondence between peptide ions and their spectra in DIA makes the identification challenging. One effective approach to reduce false positive identification is to calculate the deviation between the peptide's estimated retention time (RT) and measured RT. During this process, scaling the spectral library RT into the estimated RT, known as the RT calibration, is a prerequisite for calculating the deviation. Currently, within the DIA algorithm ecosystem, there is a lack of engine-independent and readily usable RT calibration toolkits. RESULTS: In this work, we introduce Calib-RT, a RT calibration method tailored to the characteristics of RT data. This method can achieve the nonlinear calibration across various data scales and tolerate a certain level of noise interference. Calib-RT is expected to enrich the open source DIA algorithm toolchain and assist in the development of DIA identification algorithms. AVAILABILITY AND IMPLEMENTATION: Calib-RT is released as an open source software under the MIT license and can be installed from PyPi as a python module. The source code is available on GitHub at https://github.com/chenghui03/Calib_RT.

MOTIVATION: Peptide identification of data-independent acquisition (DIA) mass spectrometry applying the peptide-centric approach heavily relies on the spectral library matching, such as the fragment intensity similarity. If the intensity similarity is calculated through all possible fragment ions of a targeted peptide instead of just a few fragment ions provided by the spectral library, the matching will be more comprehensive and reliable, and thus the identification will be more confident. In addition, the emergence of high precision spectrum predictors, like Prosit, also makes it possible to capitalize on the predicted spectrum, which contains all possible fragment ion intensities, to calculate the intensity similarity for DIA data. RESULTS: In this work, we propose Alpha-Tri, a neural-network-based model to calculate intensity similarity as a post-processing score using the predicted spectrum, measured spectrum and correlation spectrum (triple-spectrum). The predicted spectrum is generated by Prosit, the measured spectrum is retrieved from the apex of the chromatograms of all possible fragment ions and the correlation spectrum is used to indicate the present probabilities of these fragment ions as the link between the precursor and its fragment ions is lost in DIA. By adopting a data-driven method, Alpha-Tri is able to learn the intensity similarity from the triple-spectrum. This learned value is appended to initial scores from DIA-NN, allowing the ensuing statistical validation tool to report more peptides at the same false discovery rate (FDR). In our evaluation of the HeLa dataset with gradient lengths ranging from 0.5 to 2 h, Alpha-Tri delivered 3.0-7.2% gains in peptide detections at 1% FDR. On LFQbench dataset, a mixed-species dataset with known ratios, Alpha-Tri identified more peptides and proteins fell within the valid ratio ranges by up to 8.6% and 7.6%, respectively, compared with DIA-NN solely. AVAILABILITY AND IMPLEMENTATION: The original datasets for benchmarks are downloaded from the ProteomeXchange with the identifiers PXD005573, PXD000954 and PXD002952. Source code is available at https://github.com/YuAirLab/Alpha-Tri.

MOTIVATION: The peptide-centric identification methodologies of data-independent acquisition (DIA) data mainly rely on scores for the mass spectrometric signals of targeted peptides. Among these scores, the coelution scores of peak groups constructed by the chromatograms of peptide fragment ions have a significant influence on the identification. Most of the existing coelution scores are achieved by artificially designing some functions in terms of the shape similarity, retention time shift of peak groups. However, these scores cannot characterize the coelution robustly when the peak group is in the circumstance of interference. RESULTS: On the basis that the neural network is more powerful to learn the implicit features of data robustly from a large number of samples, and thus minimizing the influence of data noise, in this work, we propose Alpha-XIC, a neural network-based model to score the coelution. By learning the characteristics of the coelution of peak groups derived from the being analyzed DIA data, Alpha-XIC is capable of yielding robust coelution scores even for peak groups with interference. With this score appending to initial scores generated by the accompanying identification engine DIA-NN, the ensuing statistical validation can report the identification result and recover the misidentified peptides. In our evaluation of the HeLa dataset with gradient lengths ranging from 0.5 to 2 h, Alpha-XIC delivered 9.4-16.2% improvements in the number of identified precursors at 1% false discovery rate. Furthermore, Alpha-XIC was tested on LFQbench, a mixed-species dataset with known ratios, and increased the number of peptides and proteins fell within valid ratios by up to 16.4% and 17.8%, respectively, compared to the initial identification by DIA-NN. AVAILABILITY AND IMPLEMENTATION: Source code is available at https://github.com/YuAirLab/Alpha-XIC.