title: The influence of sample preparation and replicate analyses on HeLa Cell phosphoproteome coverage.
abstract: Ongoing optimization of proteomic methodologies seeks to improve both the coverage and confidence of protein identifications. The optimization of sample preparation, inclusion of technical replicates (repeated instrumental analysis of the same sample), and biological replicates (multiple individual samples) are crucial in proteomic studies to avoid the pitfalls associated with single point analysis and under-sampling. Phosphopeptides were isolated from HeLa cells and analyzed by nano-reversed phase liquid chromatography electrospray ionization tandem mass spectrometry (nano-RP-LC-MS/MS). We observed that a detergent-based protein extraction approach, followed with additional steps for nucleic acid removal, provided a simple alternative to the broadly used Trizol extraction. The evaluation of four technical replicates demonstrated measurement reproducibility with low percent variance in peptide responses at approximately 3%, where additional peptide identifications were made with each added technical replicate. The inclusion of six technical replicates for moderately complex protein extracts (approximately 4000 uniquely identified peptides per data set) affords the optimal collection of peptide information.
authors.affiliation: Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: Bryan M
authors.initials: BM
authors.lastname: Ham
authors.forename: Feng
authors.initials: F
authors.lastname: Yang
authors.forename: Hemalatha
authors.initials: H
authors.lastname: Jayachandran
authors.forename: Navdeep
authors.initials: N
authors.lastname: Jaitly
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Monroe
authors.forename: Marina A
authors.initials: MA
authors.lastname: Gritsenko
authors.forename: Eric A
authors.initials: EA
authors.lastname: Livesay
authors.forename: Rui
authors.initials: R
authors.lastname: Zhao
authors.forename: Samuel O
authors.initials: SO
authors.lastname: Purvine
authors.forename: Daniel
authors.initials: D
authors.lastname: Orton
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.forename: David G
authors.initials: DG
authors.lastname: Camp
authors.forename: Sandra
authors.initials: S
authors.lastname: Rossie
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D010748:Phosphopeptides / Q000032:analysis / Q000302:isolation & purification; D010750:Phosphoproteins / Q000032:analysis* / Q000302:isolation & purification; D020543:Proteome / Q000032:analysis* / Q000302:isolation & purification; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D010748:Phosphopeptides; D010750:Phosphoproteins; D020543:Proteome