Pioneer and Altimeter: Fast Analysis of DIA Proteomics Data Optimized for Narrow Isolation Windows

Advances in protein mass spectrometry have enabled a single instrument to acquire data for hundreds of samples per day, but the speed of analysis software has not kept pace. Here we introduce Pioneer and Altimeter for rapid analysis of proteomics data acquired by data-independent acquisition (DIA). Altimeter generates in silico spectral libraries, which Pioneer uses to identify and quantify peptides and proteins. Pioneer exceeds peptide coverage by up to 10%, improves quantitative accuracy, and runs four to eight times faster compared with state-of-the-art tools. Pioneer and Altimeter also model isolation-window effects on fragment isotopes to take advantage of narrow isolation windows deployed on fast-scanning time-of-flight instruments. We benchmarked Pioneer and validated its false-discovery-rate and false-transfer-rate control across diverse instruments, sample types, and acquisition settings. Altimeter and Pioneer are open source and cross-platform, with a graphical interface for streamlined analysis on personal computers and a command-line interface for compute-cluster workflows.