Bottom-up glycoproteomics by liquid chromatography–mass spectrometry (LC–MS) is an established approach for assessing glycosylation in a protein- and site-specific manner. Consequently, tools are needed to automatically align, calibrate, and integrate LC–MS glycoproteomics data. We developed a modular software package designed to tackle the individual aspects of an LC–MS experiment, called
LaCyTools. Targeted alignment is performed using user defined
m/
z and retention time (
tr) combinations. Subsequently, sum spectra are created for each user defined analyte group. Quantitation is performed on the sum spectra, where each user defined analyte can have its own
tr, minimum, and maximum charge states. Consequently, LaCyTools deals with multiple charge states, which gives an output per charge state if desired, and offers various analyte and spectra quality criteria. We compared throughput and performance of LaCyTools to combinations of available tools that deal with individual processing steps. LaCyTools yielded relative quantitation of equal precision (relative standard deviation <0.5%) and higher trueness due to the use of MS peak area instead of MS peak intensity. In conclusion, LaCyTools is an accurate automated data processing tool for high-throughput analysis of LC–MS glycoproteomics data. Released under the Apache 2.0 license, it is freely available on GitHub (
https://github.com/Tarskin/LaCyTools).