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Mass Mapping
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B065 and B017
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Protein Analytics - Mass Mapping
 

PROTOCOLS
  • Perform the digestion in a clean Eppendorf tube. 
  • To a small plug of pH-neutral polyacrylamide gel containing 100 fmol or more of purified protein, add 45 mM dithiothreitol (DTT) in 10 mM ammonium bicarbonate pH 7.8 – add a sufficient volume to cover the gel.  Incubate at 55°C for 90 minutes. 
  • Remove and discard solution by pipetting and add 100 mM Iodoacetamide – a sufficient volume to cover the gel.  Incubate at ambient temperature in the dark for 60 minutes.
  • Discard solution phase and wash the gel with 0.5 ml of 10 mM ammonium bicarbonate / 50% acetonitrile (ACN) for 30 minutes.  Discard solution phase and dry the gel to completion (covering tube loosely with foil) at ambient temperature.  At this point the dried gel can be stored at -20°C for an indefinite period.
  • Add to the gel a small volume (2 to 10 ml) of 10 mM ammonium bicarbonate containing 1 to 5 pmol trypsin.  Add a sufficient volume of 10 mM ammonium bicarbonate buffer to saturate the gel and continue adding buffer over a period of two hours until the gel is fully swollen and just covered by buffer.  Incubate at 37°C overnight.
  • Withdraw an aliquot (5% to 50%) of the solution phase.  A 1 ul aliquot may be analyzed directly in MALDI-TOF/TOF.  Alternatively, extract peptides onto Ziptips according to Millipore’s recommended protocol, wash with 0.1% trifluoroacetic acid (TFA), and elute directly to a MALDI plate using 0.5 ml 50% ACN/0.1% TFA.  Allow the eluate to dry partially, then add 0.5 ml alpha-cyano-4-hydroxycinnamic acid (CHCA, at a concentration of 5 mg/ml). 
  • Perform MALDI-MS using reflector mode to obtain monoisotopic peptide masses followed by MS/MS from selected peptides to get fragment information.
  • Search protein and genomic databases using Mascot (see reference below) or an equivalent search program.

» Reagent suppliers:
CHCA:            Agilent # G2037A
TFA:                Applied Biosystems # 400003
DTT:                Sigma # D-5545
Trypsin:            Promega Modified Trypsin # V5111
Ziptips: Micro-C18 variety: Millipore # ZTC18M096

» Remarks:
To be successful, one must prevent environmental contamination from keratin; this class of proteins can be introduced through contact with human skin or by introduction of dust present in the air.  Precautions that we take to avoid keratin contamination include filtration of solvents through microporous nylon membranes, use of pipet tips and Eppendorf tubes that are certified DNAse/RNAse-free, use of powder-free gloves, and by avoidance of unnecessary exposure of the samples to air.
We use a Sciex TOF/TOF 5800 MALDI mass spectrometer in MS mode, typically accurate to 40 ppm, and routinely obtain up to fifteen MS/MS spectra from selected peptide ions. 
The mass map obtained is used to match the protein to theoretical digests archived in a variety of databases (typically NCBInr).  Mascot MS/MS ions search, (http://www.matrixscience.com/search_form_select.html), or other similar search programs should be consulted for additional information.