title: Ultrasensitive proteomics using high-efficiency on-line micro-SPE-nanoLC-nanoESI MS and MS/MS.
abstract: Ultrasensitive nanoscale proteomics approaches for characterizing proteins from complex proteomic samples of <50 ng of total mass are described. Protein identifications from 0.5 pg of whole proteome extracts were enabled by ultrahigh sensitivity (<75 zmol for individual proteins) achieved using high-efficiency (peak capacities of approximately 10(3)) 15-microm-i.d. capillary liquid chromatography separations (i.e., using nanoLC, approximately 20 nL/min mobile-phase flow rate at the optimal linear velocity of approximately 0.2 cm/s) coupled on-line with a micro-solid-phase sample extraction and a nanoscale electrospray ionization interface to a 11.4-T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (MS). Proteome measurement coverage improved as sample size was increased from as little as 0.5 pg of sample. It was found that a 2.5-ng sample provided 14% coverage of all annotated open reading frames for the microorganism Deinococcus radiodurans, consistent with previous results for a specific culture condition. The estimated detection dynamic range for detected proteins was 10(5)-10(6). An improved accurate mass and LC elution time two-dimensional data analysis methodology, used to both speed and increase the confidence of peptide/protein identifications, enabled identification of 872 proteins/run from a single 3-h nanoLC/FTICR MS analysis. The low-zeptomole-level sensitivity provides a basis for extending proteomics studies to smaller cell populations and potentially to a single mammalian cell. Application with ion trap MS/MS instrumentation allowed protein identification from 50 pg (total mass) of proteomic samples (i.e., approximately 100 times larger than FTICR MS), corresponding to a sensitivity of approximately 7 amol for individual proteins. Compared with single-stage FTICR measurements, ion trap MS/MS provided a much lower proteome measurement coverage and dynamic range for a given analysis time and sample quantity.
authors.affiliation: Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Yufeng
authors.initials: Y
authors.lastname: Shen
authors.forename: Nikola
authors.initials: N
authors.lastname: Tolić
authors.forename: Christophe
authors.initials: C
authors.lastname: Masselon
authors.forename: Ljiljana
authors.initials: L
authors.lastname: Pasa-Tolić
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Kim K
authors.initials: KK
authors.lastname: Hixson
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Gordon A
authors.initials: GA
authors.lastname: Anderson
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification; D034301:Deinococcus / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D036103:Nanotechnology / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
chemical_list: D001426:Bacterial Proteins; D012710:Serum Albumin, Bovine