In-Gel Digestion

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In-Gel Digestion Why In-Gel Digest? Difficult
/ impossible to extract intact proteins from the
gel Generate peptides that facilitate Protein
ID Enzymes? Trypsin, Trypsin,
Trypsin--Cleaves after Arg (R) Lys (K) Lys
C--Cleaves after Lys (K) V8-- (Cleaves after
Asp (D) Glu (E) Reduction/Alkylation of Cys
(C)? Necessary for 1D bands and Silver Stained
2D gel plugs Optional for CBB or Sypro stained
2D gel plugs
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The Steps of an In-Gel Digestion
Wash, wash, wash alternating 25mM Ammonium
Bicarbonate (ABC) and Acetonitrile (ACN) Why?
to remove residual SDS acetic
acid Reduction/Alkylation Reduce with
DTT Alkylate with Iodoactamide Why? ...to
recover Cys containing peptides Wash
Again Dehydrate with ACN / speedvac
Rehydrate/Incubate with Trypsin Extract
peptides (ACN/Formic Acid) De-salt/concentrate
(ZipTips) spot to a MALDI target
ProGest
ProMS
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http//www.genomicsolutions.co.uk/software/
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Protein Identification
Peptide Mass Fingerprint (PMF) Measure the
mass of each tryptic peptide Query a database
of known proteins This can be either a protein
database or a DNA database that undergoes a 6
frame translation See how many peptides from
the experimental data match to theoretical
tryptic peptides of proteins in the
database Most commonly used database search
engines are Protein Prospector
http//prospector.ucsf.edu Mascot http
//www.matrixscience.com
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MALDI Matrix-Assisted Laser Desorption
Ionization
Advantages Rapid analysis/high
throughput Acquisition is easily
automated Disadvantages Sensitivity not as
good as LC/MS/MS Many (5-7) peptides
required for a confident ID Protein must be in
the database to be identified Unable to query
EST databases No sequence data is obtained
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When to Use MALDI for Protein ID
CBB stained 2D gel plug (Success rate
99) Silver Stained gel plug (Success rate
50-60 ) the success rate increases as the
protein amount increases Bands from SDS Page
gels (and many 2D features) contain more than
one protein, MALDI will ID only the 1-3 most
abundant proteins. Protein ID by MALDI
(peptide mass fingerprint) is only successful if
the protein is in a protein database or DNA
database (Most useful when the genome of the
organism is known.)
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What is LC/MS/MS?
The digested pool of peptides is injected onto a
reverse phase C18 column. (300 mm to 50mm
diameter) As the peptides elute from the column
instead of being detected by a UV detector they
are directly infused into an Electrospray
Ionization (ESI) mass spectrometer. The tandem
mass spectrometer (Triple Quadrupole, Ion Trap,
or Quadrupole TOF) measures the m/z of the
peptides as they elute, selects a single m/z,
then fragments that peptide and measures the m/z
of all the fragment ions.
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Protein Identification Using Fragment Ion Data
The mass of each peptide along with the mass of
the fragment ions is used to query the
database A single peptide can provide a
confident ID Because of this many proteins can
be identified in a mixture. The data can be
searched against EST databases If no protein is
identified then the data can be
manually interpreted to get the full length
sequence of the peptide (de novo
sequencing) The sequence data can be used to map
post-translational modifications
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LC/MS/MS
Advantages Can be the most sensitive method for
protein ID (the smaller the column the more
sensitive) Unambiguous search results (very few
false positives) Only a single peptide is
required for ID The ability to perform limited
homology searches single substitution within a
peptide Able to ID proteins from simple mixtures
(20) such as immunoprecipitates The data
can be searched against EST databases
Disadvantages Time (Both acquisition and
analysis) Chromatography problems of NanoLC