title: Automated integration of monolith-based protein separation with on-plate digestion for mass spectrometric analysis of esophageal adenocarcinoma human epithelial samples.
abstract: A unique approach of automating the integration of monolithic capillary HPLC-based protein separation and on-plate digestion for subsequent MALDI-MS analysis has been developed. All liquid-handling procedures were performed using a robotic module. This automated high-throughput method minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and analysis. Also, precise positioning of the droplet from the capillary HPLC separation onto the MALDI plate allows for preconcentration effects of analytes for improved sensitivity. Proteins from primary esophageal Barrett's adenocarcinoma tissue were prefractionated by chromatofocusing and analyzed successfully by this automated configuration, obtaining rapid protein identifications through PMF and sequencing analyses with high sequence coverage. Additionally, intact protein molecular weight values were obtained as a means to further confirm protein identification and also to identify potential sequence modifications of proteins. This simple and rapid method is a highly versatile and robust approach for the analysis of complex proteomes.
authors.affiliation: Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Chul
authors.initials: C
authors.lastname: Yoo
authors.forename: Jia
authors.initials: J
authors.lastname: Zhao
authors.forename: Manoj
authors.initials: M
authors.lastname: Pal
authors.forename: Katherine
authors.initials: K
authors.lastname: Hersberger
authors.forename: Christian G
authors.initials: CG
authors.lastname: Huber
authors.forename: Diane M
authors.initials: DM
authors.lastname: Simeone
authors.forename: David G
authors.initials: DG
authors.lastname: Beer
authors.forename: David M
authors.initials: DM
authors.lastname: Lubman
mesh_terms: D000230:Adenocarcinoma / Q000378:metabolism*; D001471:Barrett Esophagus / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004938:Esophageal Neoplasms / Q000378:metabolism*; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D009363:Neoplasm Proteins; D020543:Proteome