Identification and quantification of large numbers of proteins is important for the characterization of biological systems to gain insight into their composition and function. The ZenoTOF 7600 system is equipped with a Zeno trap that improves the duty cycle to more than 90% at the MS/MS level, enabling gains in sensitivity of 5 to 20x. Improved MS/MS sensitivity is important for single-cell and other applications in which samples are present at low-nanogram levels, as it enables more peptide and protein identifications. Here, we evaluated the performance of data-dependent acquisition (DDA) up to 500 ng of commercial digest loads and data-independent acquisition (DIA) approaches on a novel ZenoTOF 7600 system by testing loads ranging from 0.25 ng to 50 ng.
We used a ZenoTOF 7600 system with Zeno SWATH acquisition enabled in-line with a Waters M-Class LC system to determine protein identifications across varying commercial K562 tryptic digest loads. We determined protein identifications in either DDA or Zeno SWATH acquisition modes.
We used the Zeno SWATH acquisition capability with a 45-min gradient to test cell digest loads that were within the single-cell regime, such as 0.25, 0.5 and 1 ng loads. From these experiments, over 1000-1300, 1400 and 2300 protein groups were identified, for 0.25, 0.5 and 1 ng loads, respectively, and 45-55% of these identifications had a CV less than 20% when searched against a spectral library. At the precursor level for the same loads, there were 3000-4000, 5500 and 12000 corresponding precursors for the 0.25, 0.5 and 1 ng loads. We tested higher loads and identified 4200, 5000 and 6100 protein groups for 5, 10 and 25 ng loads, respectively, and 64-83% of these identifications satisfied the 20% CV cutoff. For a 50 ng load, more than 6300 protein groups were identified, of which 90% had less than 20% CV, and 56000 precursors were identified. When the data were searched against a FASTA library in library-free mode, the overall number of identifications and those at 20% CV cutoffs approach those achieved when processed using the spectral library approach.
A 200 ng and 500 ng load of K562 tryptic digest was tested in DDA mode on a Waters Acquity column, using a 180-min gradient. From these experiments, we were able to identify more than 4600 and 5000 protein groups for the two loads, respectively, with over 40000 and 50000 peptides for each load.