Use of captive spray ionization to increase throughput of the data-independent acquisition technique PAcIFIC

Rationale: The Precursor Acquisition Independent From Ion Count (PAcIFIC) method is a data-independent acquisition technique capable of identifying proteins over eight orders of magnitude in a single analysis in human plasma. Widespread application of this technique in proteomic studies is hindered by its time-intensive nature. There exists a need to explore strategies to increase the throughput of the PAcIFIC method.

Methods: The PAcIFIC acquisition technique was optimized for use with an Orbitrap mass spectrometer fitted with a captive spray ionization (CSI) source. Chromatographic methods, PAcIFIC acquisition parameters, for example, channels interrogated per chromatographic gradient, time span of chromatographic gradient, and sample loading amount, were investigated to achieve a maximum number of peptide and protein identifications on a yeast proteome where protein copy number had been previously determined.

Results: A 24-hour CSI PAcIFIC method was developed with minimal reduction of peptide and protein identifications from the 4.2-day nano-electrospray ionization (nESI) PAcIFIC method. Analysis of a yeast cell lysate with the 4.2-day nESI PAcIFIC method resulted in 13,468 peptide and 2120 protein identifications. A 24-hour CSI PAcIFIC method resulted in 11,277 peptide and 1753 protein identifications. Increased sample loading of the CSI system allowed for an 8% increase in peptide and protein identifications.

Conclusions: A dramatic decrease in the overall analysis time of the PAcIFIC method (24 h with CSI versus 100.8 h with nESI) was achieved with minimal reduction of peptide and protein identifications. Furthermore, the CSI PAcIFIC method demonstrated a high degree of sensitivity and capability of identifying proteins across a large dynamic range observed with the nESI PAcIFIC method (CSI PAcIFIC identified proteins as low as 46 molecules per cell).