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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)

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In-Gel Digestion Why In-Gel Digest? Difficult / impossible to extract intact proteins from the gel

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  1. 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

  2. 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

  3. http://www.genomicsolutions.co.uk/software/

  4. 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

  5. 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

  6. 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.)

  7. 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.

  8. 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

  9. 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

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