title: An optimized matrix-assisted laser desorption/ionization sample preparation using a liquid matrix, 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid, for phosphopeptides.
abstract: BACKGROUND
  A liquid matrix, 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA), introduced by Kolli et al. in 1996 for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported for peptides and proteins, oligonucleotides, oligosaccharides, and glycopeptides. However, it has not been validated for phosphopeptides.
  
  
  METHODS
  We optimized sample preparation using 3-AQ/CHCA for phosphopeptides. The sensitivity of six phosphopeptide species as isolated or in digests was systematically evaluated by using MALDI-quadropole ion trap (QIT)-time of flight (TOF) MS in positive and negative ion modes, and compared with the conventional methods using a solid matrix, 2,5-dihydroxybenzoic acid (2,5-DHB).
  
  
  RESULTS
  The sensitivity of mono- and tetraphosphopeptides was improved 10- to 10 000-fold with the optimized preparation method using 3-AQ/CHCA compared with the conventional methods using 2,5-DHB. Improvement by 3-AQ/CHCA itself was 10-fold. Adding ammonium dihydrogen phosphate or an analyte solvent composition was also effectively improved the sensitivity. Phosphopeptides in isolated form or in digests were detected at femto- or subfemtomole levels.
  
  
  CONCLUSIONS
  Sensitivity of phosphopeptides was improved by the optimized sample preparation method using 3-AQ/CHCA compared with the conventional method using 2,5-DHB. The validation of 3-AQ/CHCA for phosphopeptides was systematically confirmed, expanding the potential of this matrix to phosphoproteomics.
authors.affiliation: Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1 Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto, 604-8511, Japan. yukof@shimadzu.co.jp
authors.forename: Yuko
authors.initials: Y
authors.lastname: Fukuyama
authors.forename: Kohei
authors.initials: K
authors.lastname: Takeyama
authors.forename: Shin-ichirou
authors.initials: S
authors.lastname: Kawabata
authors.forename: Shinichi
authors.initials: S
authors.lastname: Iwamoto
authors.forename: Koichi
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000634:Aminoquinolines / Q000737:chemistry*; D000818:Animals; D002364:Caseins / Q000737:chemistry; D002645:Chickens; D003373:Coumaric Acids / Q000737:chemistry*; D008969:Molecular Sequence Data; D010047:Ovalbumin / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D011972:Receptor, Insulin / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000634:Aminoquinolines; D002364:Caseins; D003373:Coumaric Acids; D010748:Phosphopeptides; C007175:alpha-cyano-4-hydroxycinnamate; D010047:Ovalbumin; D011972:Receptor, Insulin