Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry.
A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.
Lim
Hanjo
H
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Eng
Jimmy
J
Yates
John R
JR
Tollaksen
Sandra L
SL
Giometti
Carol S
CS
Holden
James F
JF
Adams
Michael W W
MW
Reich
Claudia I
CI
Olsen
Gary J
GJ
Hays
Lara G
LG
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D030562:Databases, Protein; D015180:Electrophoresis, Gel, Two-Dimensional; D017017:Methanococcus / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D020027:Pyrococcus furiosus / Q000737:chemistry; D012680:Sensitivity and Specificity; D012984:Software; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin
D011506:Proteins; D014357:Trypsin