In-Gel Digestion Why In-Gel Digest? Difficult / impossible to extract intact proteins from the gel
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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
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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) - PowerPoint PPT Presentation
Transcript of In-Gel Digestion Why In-Gel Digest? Difficult / impossible to extract intact proteins from the gel
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 plugsOptional for CBB or Sypro stained 2D gel plugs
Wash, wash, wash…
alternating 25mM Ammonium Bicarbonate (ABC) and Acetonitrile (ACN)Why? …to remove residual SDS & acetic acid
Reduction/Alkylation Reduce with DTTAlkylate with IodoactamideWhy? ...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
The Steps of an In-Gel Digestion
ProGest
ProMS
http://www.genomicsolutions.co.uk/software/
Protein Identification
Peptide Mass Fingerprint (PMF)
Measure the mass of each tryptic peptide
Query a database of known proteinsThis can be either a protein database ora 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
Mascothttp://www.matrixscience.com
AdvantagesRapid analysis/high throughputAcquisition is easily automated
DisadvantagesSensitivity not as good as LC/MS/MS
Many (~5-7) peptides required for a confident IDProtein must be in the database to be identifiedUnable to query EST databases No sequence data is obtained
MALDIMatrix-Assisted Laser Desorption Ionization
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.)
When to Use MALDI for Protein ID
What is LC/MS/MS?
The digested pool of peptides is injected onto a reverse phase C18 column. (300 m to 50m 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, IonTrap, 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.
Protein Identification Using Fragment Ion Data
The mass of each peptide along with the mass of the fragmentions is used to query the database
A single peptide can provide a confident IDBecause 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 manuallyinterpreted to get the full length sequence of the peptide(de novo sequencing)
The sequence data can be used to map post-translational modifications
LC/MS/MS
AdvantagesCan 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 IDThe ability to perform limited homology searches
single substitution within a peptideAble to ID proteins from simple mixtures (~20)
such as immunoprecipitates The data can be searched against EST databases
DisadvantagesTime (Both acquisition and analysis)Chromatography problems of NanoLC
