LAUSR

Rationale Analyte quantitation by mass spectrometry underpins a diverse range of scientific endeavors. The fast‐growing field of mass spectrometer development has resulted in several targeted and untargeted acquisition modes suitable for these applications. By characterizing the acquisition methods available on an ion mobility (IM)‐enabled orthogonal acceleration time‐of‐flight (oa‐ToF) instrument, the optimum modes for analyte semi‐quantitation can be deduced. Methods Serial dilutions of commercial metabolite, peptide, or cross‐linked peptide analytes were prepared in matrices of human urine or Escherichia coli digest. Each analyte dilution was introduced into an IM separation‐enabled oa‐ToF mass spectrometer by reversed‐phase liquid chromatography and electrospray ionization. Data were acquired for each sample in duplicate using nine different acquisition modes, including four IM‐enabled acquisitions modes, available on the mass spectrometer. Results Five (metabolite) or seven (peptide/cross‐linked peptide) point calibration curves were prepared for analytes across each of the acquisition modes. A nonlinear response was observed at high concentrations for some modes, attributed to saturation effects. Two correction methods, one MS1 isotope‐correction and one MS2 ion intensity‐correction, were applied to address this observation, resulting in an up to twofold increase in dynamic range. By averaging the semi‐quantitative results across analyte classes, two parameters, linear dynamic range (LDR) and lower limit of quantification (LLOQ), were determined to evaluate each mode. Conclusion A comparison of the acquisition modes revealed that data‐independent acquisition and parallel reaction monitoring methods are most robust for semi‐quantitation when considering achievable LDR and LLOQ. IM‐enabled modes exhibited sensitivity increases, but a simultaneous reduction in dynamic range required correction methods to recover. These findings will assist users in identifying the optimum acquisition mode for their analyte quantitation needs, supporting a diverse range of applications and providing guidance for future acquisition mode developments.