KSEF KCF:ProteoTAG Systems

  • Mohan, Royce (PI)
  • Kim, Kyung (CoI)

Grants and Contracts Details


Billerica, MA) operated in the reflectron mode will be used to generate peptide mass fingerprints (PMFs). PeBtides resulting from in-gel digestion will be spotted on to a 384 position, 600 IJm AnchorChipT Target (Bruker Daltonic, Bremen, Germany) and prepared according to AnchorChip recommendations. Briefly, 1 IJL of digestate will be mixed with 1 IJL of alpha-cyano- 4-hydoxycinnamic acid (0.3 mg/mL in ethanol:acetone 2:1) directly on the target and allowed to dry at room temperature. The sample spot will be washed with 1 IJL of a 1% TFA solution for approximately 60 seconds. The TFA droplet will be gently separated from the sample spot by a stream of compressed air. The resulting diffuse sample spot will be recrystallized (refocused) using 1 IJL of a solution of ethanol:acetone:0.1 % TFA (6:3: 1). Reported spectra typically result from the summation of 100 laser shots. External calibration of the mass axis will be used for acquisition and internal calibration using either trypsin autolysis ions or matrix clusters will be applied post acquisition for accurate mass determination. All PMFs are expected to have a mass accuracy of at least 150 ppm. Non-compliant samples will be re-spotted. LC/MS/MS spectra from simple systems (gel spots) will be acquired on a Finnigan LCa "Classic" quadrupole ion trap mass spectrometer. Separations will be performed with an HP 1100 HPLC modified with a custom splitter to deliver 4 IJUmin to a C18 capillary column (300 IJm id x 15 cm), packed in-house with Macrophere 300 5IJm C18 (Alltech Associates, Deerfield, IL). Gradient separations initiate at 95% water:5% acetonitrile (both phases contain 0.1% formic acid) for 2 min, increase to 20% acetonitrile over 8 min, then increase to 90% acetonitrile over 25 min, and are held at 90% acetonitrile for 8 min, then increase to 95% in 2 min, and finally return to initial conditions in 10 min (total time 45 min with a 10 min recycle time). Tandem mass spectra will be acquired in a data dependent manner. Three microscans are typically averaged to generate the data dependent full scan spectrum with the most intense ion subjected to tandem mass spectrometry where typically five microscans are averaged to produce the MS/MS spectrum. Masses subjected to the MS/MS scan are excluded from further mass spectrometry for 2 minutes. All mass spectral data (PMFs and MS/MS data) will be searched against the NCBI nonredundant database (updated monthly) using the MASCOT search algorithm.
Effective start/end date4/1/079/30/10


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