Abstract
The effects of MS1 injection time, MS2 injection time, dynamic exclusion time, intensity threshold, and isolation width were investigated on the numbers of peptide and protein identifications for single-shot bottom-up proteomics analysis using CZE-MS/MS analysis of a Xenopus laevis tryptic digest. An electrokinetically pumped nanospray interface was used to couple a linear-polyacrylamide coated capillary to a Q Exactive HF mass spectrometer. A sensitive method that used a 1.4 Th isolation width, 60,000 MS2 resolution, 110 ms MS2 injection time, and a top 7 fragmentation produced the largest number of identifications when the CZE loading amount was less than 100 ng. A programmable autogain control method (pAGC) that used a 1.4 Th isolation width, 15,000 MS2 resolution, 110 ms MS2 injection time, and top 10 fragmentation produced the largest number of identifications for CZE loading amounts greater than 100 ng; 7218 unique peptides and 1653 protein groups were identified from 200 ng by using the pAGC method. The effect of mass spectrometer conditions on the performance of UPLC-MS/MS was also investigated. A fast method that used a 1.4 Th isolation width, 30,000 MS2 resolution, 45 ms MS2 injection time, and top 12 fragmentation produced the largest number of identifications for 200 ng UPLC loading amount (6025 unique peptides and 1501 protein groups). This is the first report where the identification number for CZE surpasses that of the UPLC at the 200 ng loading level. However, more peptides (11476) and protein groups (2378) were identified by using UPLC-MS/MS when the sample loading amount was increased to 2 μg with the fast method. To exploit the fast scan speed of the Q-Exactive HF mass spectrometer, higher sample loading amounts are required for single-shot bottom-up proteomics analysis using CZE-MS/MS.