Refining nanoflow LC and orbitrap MS data acquisition parameters for pico- and nanogram scale proteomics

Researchers need enhanced analytical techniques to profile and characterize tissue and cellular proteomes in studying nanogram scale peptide samples. To meet this demand, nanoflow liquid chromatography (nLC) and mass spectrometry (MS) work has focused on method development, while improvements are made in new cell sorting and isolation instrumentation. In this article, we describe improvements in peptide and protein identifications using simple, cost-effective changes to common mass spectrometry procedures. We focused on procedures that used an Orbitrap instrument to analyze 1 nanogram of peptide material or less. We found protein identifications increased over 40% when applying lowered precursor intensity thresholds in data-dependent selection. We also demonstrate improvements in identifying late-eluting peptides using sample diluents containing <i>n</i>-dodecyl-β-D-maltoside (DDM). We also show lower nLC flow rates can enhance protein identifications over 20%. Finally, we report improvements of 18% in peptide identifications when multiple high-field asymmetric waveform ion mobility spectrometry (FAIMS) compensation voltages (CV) are applied within a single method. These simple modifications provide researchers with options to improve peptide detection in very limited or low concentration samples.