title: Use of captive spray ionization to increase throughput of the data-independent acquisition technique PAcIFIC.
abstract: BACKGROUND
  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).
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA, USA.
authors.forename: John D
authors.initials: JD
authors.lastname: Chapman
authors.affiliation: Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA, USA.
authors.forename: J Scott
authors.initials: JS
authors.lastname: Edgar
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD, USA.
authors.forename: Young Ah
authors.initials: YA
authors.lastname: Goo
mesh_terms: D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015003:Yeasts / Q000737:chemistry* / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D010455:Peptides