Mass spectrometric protein identification
In-gel digestion was performed mainly as previously described [12] with some modifications
[10]. Extracted peptides were used for MALDI-TOF analysis. Mass spectrometric analysis of
peptide mass fingerprinting (PMF) was performed using a Voyager-DE STR MALDI-TOF-MS
(PerSeptive Biosystems, Framingham, MA, USA). Approximately 1 L of extracted peptide
solution from each gel spot piece and the same volume of matrix solution (10 mg/mL -ciano-4hydroxycinnamic acid, 0.1% v/v TFA, and 50% v/v acetonitrile) were loaded onto a MALDI
sample plate (96 well) and crystallized. After MALDI-TOF, Peak assignment was performed
manually using DataExplorerTM software that is part of the Voyager-DE STR MALDI-TOF-MS
software package (PerSeptive Biosystems, Framingham, MA, USA) and spectra were saved as
peak table files (*.pkt) to search against non-redundant protein sequence database on the internet
(SWISS-PROT, IPI COW and/or NCBInr (2009/5/29) Data Bank). The PMF data were applied to
ProFound and MASCOT search engines (http://prowl.rockefeller.edu/prowl-cgi/profound.exe and
http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF).
Search
parameters were other mammalia as taxonomy, trypsin as an enzyme, carbamidomethylation of
cystein as fixed modification and methionine oxidation as variable modification, and one missed
cleavage site with a mass tolerance of less then 500 ppm. The protein identification was further
validated by ProFound and MASCOT score, sequence coverage and number of peptides matched
versus number submitted. Also, for LC-MS/MS, resulting tryptic peptides were separated and
analyzed using reversed phase capillary HPLC directly coupled to a Finnigan LCQ ion trap mass
spectrometer (LC-MS/MS). Both of a 0.1 ⅹ 20 mm trapping and a 0.075 ⅹ 130 mm resolving
column were packed with Vydac 218 MS low trifluoroactic acid C18 beads (5㎛ in size, 300 Å in
pore size; Vydac, Hesperia, CA, USA) and placed in-line. Following the peptides were bound to
the trapping column for 10 min at with 5% (v/v) aqueous acetonitrile containing 0.1% (v/v)
formic acid, then the bound peptides were eluted with a 50-min gradient of 5 – 80% (v/v)
acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.2㎕/min. For tandem mass
spectrometry, a full mass scan range mode was m/z = 450 – 2000Da. After determination of the
charge states of an ion on zoom scans, product ion spectra were acquired in MS/MS mode with
relative collision energy of 55%.
The individual spectra from MS/MS were processed using the TurboSEQUEST software
(Thermo Quest, San Jose, CA). The generated peak list files were used to query either MSDB
database or NCBI using the MASCOT program. Only significant hits as defined by MASCOT
probability analysis were considered initially.