Advances in the online separation of complex proteomics samples including ultra-high performance liquid chromatography (UHPLC), ultra-high resolution separation columns, and high-field asymmetric waveform ion mobility spectrometry (FAIMS) enable a deeper mining of the proteome with single-shot methods. In addition to the improved separation of complex proteomes with single-shot methods described above, the CHIMERYS™ intelligent search algorithm unlocks the ability to deconvolute the chimeric spectra that still arise from the co-isolation and fragmentation of multiple peptides in tandem mass spectra from both Orbitrap and ion trap mass analyzers.
HeLa protein digests were resuspended in 5% ACN / 0.1% FA, and then injected via an autosampler onto analytical columns, and separated at 300 nL/min using a Thermo Scientific™ Vanquish™ Neo UHPLC system. Data were collected using a Thermo Scientific™ FAIMS Pro Duo™ interface and Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer in data-dependent acquisition mode. Data files were processed with Thermo Scientific™ Proteome Discoverer™ 3.0 software.
To optimize the acquisition strategy for single-shot performance, 1 µg HeLa digest and a 1-hour gradient were used with isolation widths between 0.4 and 3 Th. Using an isolation width of 1.5 Th resulted in an average of 7,814 proteins, 59,213 peptides, and 128,647 peptide spectrum matches (PSMs) per run, increases of 13%, 31%, and 29% compared to a 0.4 Th isolation window, respectively. The use of a wider isolation window was enabled by the deconvolution capabilities of the CHIMERYS workflow, which provided 1.02 PSMs per spectra on average with the 1.5 Th isolation window versus 0.79 PSMs per spectra for the 0.4 Th isolation window. In contrast, processing the 1.5 Th isolation window results using a Sequest HT workflow provided an average of only 6,511 proteins, 39,549 peptides, and 53,289 PSMs. Thus, CHIMERYS provided improvements of 20% for proteins, 50% for peptides, and 141% for PSMs. To determine the impact of the increased isolation window on the ability to perform deeper analysis of longer gradient single-shot proteomes, 1 µg of HeLa were run using a 2-hour and 3-hour gradient. The 2 hour runs identified an average of 8,424 proteins, 76,619 peptides, and 187,391 PSMs, while the 3 hour runs identified 8,731 proteins, 84,084 peptides, and 248,076 PSMs. These results demonstrate that coupling advanced capabilities in online separation to enhanced data acquisition and intelligent data processing allows for substantial improvements in single-shot proteomics performance to yield a more thorough coverage of biological pathways with higher throughput.