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LC MS/MS

For protein sequencing by tandem MS (or LC/MS/MS) the protein would be digested into peptides and spectra obtained for the major peptide ions.

Data analysis falls into two types :

1.The sample spectra is compared to databases to look for identical sequences. This can be adequate to identify a known protein, or closely related proteins.
2.The sample spectra is manually interpreted to obtain de novo protein sequence for each peptide ion. This is the equivalent of N-terminal Edman sequencing of internal peptides and, if successful gives the highest quality data. The sequences can range in length from 6-20+ amino acids and these can be used to design a probe (the identification of several peptides may be required to produce a sequence that best suits design of an oligo probe).
We recommend 3 peptides are sequenced per sample.

Sample Preparation
The protein can be sent as a band or spot excised from an SDS-gel (either stained with Coomassie or MS compatible silver stain). The gel piece should be air dried in a microcentrifuge tube. Care must be taken to ensure no contamination of the bands or spots with human proteins (skin) or dust. PVDF membrane cannot be analyzed by mass spectrometry. Sample can be analyzed in liquid form. The sample should be pure and it must be freeze dried or air dried in a micro-centrifuge tube prior to shipping.

Amount of protein required
For a pure protein 100ng-1ug.

PEPTIDE MASS FINGERPRINTING BY MALDI-TOF

This method uses the protein directly from an SDS-PAGE gel. The band or spot can be stained with coomassie and excised into a small sample tube and dried. The protein is then relatively stable and can be sent by post/courier. Most bands visible by Coomassie staining will produce identification. Protein identification is also possible from silver or sypro stained proteins.

N-TERMINAL SEQUENCING

This is the state of the art technique for obtaining internal sequence information, and with an appropriate strategy can be used to sequence entire proteins. This technique can readily provide sufficient data to show homology with other proteins, or for novel proteins to obtain internal amino acid sequence information to enable cloning of the gene. The alternative approach of traditional N-terminal Edman sequencing is less sensitive and slightly more expensive. It has the benefit of providing the definitive sequence of the N-terminus.

Sample preparation (from gels)
The protein should be electrophoretically transferred to PVDF membrane. The membrane should be stained with Coomassie Blue, and the target band must then be visible by eye. The entire membrane should be sent intact along with a photocopy indicating the band to be sequenced. Sample can be analyzed from liquid form. However the sample must be freeze dried in a micro-centrifuge tube prior to shipping.

Amount of protein required
For a pure protein the sensitivity limit is approximately 1 picomole, however we recommend 10pmol to ensure a satisfactory result.

AMINO ACID ANALYSIS

Chemical hydrolysis of protein and peptide samples - ideal for quality control.

Sample Preparation
The sample should contain 5 micrograms or more of the proteins to be analyzed. The sample should be lyophilized before shipment

2 DIMENSIONAL GEL ELECTROPHORESIS

Images are analyzed using state of the art pattern recognition software (Progenesis) to ensure the most accurate interpretation of protein spot data

The sample should be in the form of a crude protein extract pellet.

Amount
1-2mg total protein (assuming a complex protein mixture) is required for a large format (20cm) gel stained with Coomassie.
0.3-0.5 mg total protein (assuming a complex protein mixture) is required for a small format (12cm) gel stained with Coomassie.

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