BrIS: Visualizing Internal Standards at the MS1 Level via Bromine Fingerprint for Sensitive and Robust Proteomics...

Data fidelity in mass spectrometry (MS)-based proteomics demands stringent quality control, for which spiking internal standards into biological samples is a straightforward practice. Yet, conventional internal-standard tracking relies on MS/MS-dependent identification, which can be unavailable or unreliable under challenging analytical conditions. Herein, we introduce a suite of Brominated Internal Standards (BrIS) and a tailored scanning algorithm, BrScan-MS1, which exploits bromine isotopic fingerprints and conserved elution order of BrIS peptides to directly track them at the MS1 level and enable reliable retention time (RT) assignment. Across a DDA dilution series, BrScan-MS1 recognized all BrIS peptides in every BrIS-spiked replicate and provided consistent RT assignments. In long-term DIA analyses using a plasma matrix, BrIS showed greater RT stability and quantitative consistency than iRT peptides and enabled sensitive tracking of instrumental drift. Cross-platform analyses demonstrated robust BrIS-based RT calibration of endogenous peptides across LC-MS platforms. In single-cell proteomics involving three cell lines, BrIS enabled reliable tracking at low sample inputs while largely maintaining cell-line-specific proteomic patterns and showed a modest tendency to better preserve control-derived differential-expression patterns than iRT peptides. Together, BrIS and BrScan-MS1 provide a practical approach for quality control across diverse proteomics settings ranging from complex matrices to ultra-low-input samples.