title: Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry.
abstract: Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases-casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6-along with16O4- and18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. Graphical Abstract ᅟ.
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.
authors.forename: Chuan-Chih
authors.initials: CC
authors.lastname: Hsu
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.|Celgene Corporation, Cambridge, MA, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Xue
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.
authors.forename: Justine V
authors.initials: JV
authors.lastname: Arrington
authors.affiliation: Department of Horticulture and Landscape, Purdue University, West Lafayette, IN, 47907, USA.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wang
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.
authors.forename: Juan Sebastian
authors.initials: JS
authors.lastname: Paez Paez
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: Department of Horticulture and Landscape, Purdue University, West Lafayette, IN, 47907, USA.
authors.forename: Jian-Kang
authors.initials: JK
authors.lastname: Zhu
authors.affiliation: Department of Biochemistry, Purdue University, West Lafayette, IN, 47907, USA. watao@purdue.edu.
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry; D029681:Arabidopsis Proteins / Q000032:analysis / Q000737:chemistry; D047390:Casein Kinase II / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods; D007553:Isotope Labeling; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism; D010103:Oxygen Isotopes / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D010936:Plant Extracts / Q000032:analysis / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D029681:Arabidopsis Proteins; D010103:Oxygen Isotopes; D010748:Phosphopeptides; D010936:Plant Extracts; D000255:Adenosine Triphosphate; D047390:Casein Kinase II; D020928:Mitogen-Activated Protein Kinases
keywords: Protein phosphorylation; Proteomics; Tandem mass spectrometry