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Identify single proteins by MALDI MS

Identification of single protein excised from 1D and 2D gels

Would you like to identify the single most abundant protein present in a 1D gel band or 2D gel spot? Just send us the gel band/spot and we’ll identify this protein by its mass spectrometric peptide fingerprint. Trypsin in-gel digestion is followed by MALDI MS and MALDI MS-MS analysis of the most abundant peptides. We search your data against a public protein database from UniProt. This database contain amino acid sequences for more than 80 million proteins. Each month the databases are updated to consider all known proteins.

Choose your service

Our PICK ‘n POST™ services are fast protein analysis without customization. You get quality results in easy to understand standard reports, at fixed prices.

Option 1

ID of single protein by MALDI-MS/MS (in-gel)

  • In-gel trypsin digest, peptide analysis by MALDI-MS/MS
  • Search against UNIPROT database, ID of single protein in sample
  • Sequence coverage map for identified protein
$225.00
Per sample
Results in 4-8 days
Service includes
  • You provide us with gel band/spot for analysis and we perform in-gel reduction/alkylation and trypsin digest.
  • We run a MALDI MS and MS/MS analysis.
  • This is followed by a MASCOT search in NCBI/UniProt protein database.
  • You obtain identification of single proteins for each sample.
  • The analysis report is delivered within 4-8 days.

Results from protein identification database search

Applications
Our customers typically use this analysis for the following applications:

Identify the single most abundant protein present in a 1D gel band or 2D gel spot

Verification of correct protein expression

Identification of unknown proteins

Identification of degradation products

Sample requirements

You can prepare the gel bands/spots for this analysis in two ways, either by 1D SDS PAGE or by 2D gels.

Stain the gel with Coomassie Blue, SYPRO Ruby or Silver stain. It is essential to use a staining protocol or kit that has been optimized for MS analysis. We recommend using one of the following kits:

  • ProteoSilver Plus, Sigma (Product # PROTSIL1 or PROTSIL2)
  • Dodeca Silver Stain, BioRad (Product # 161-0481 or 161-0480)
  • Or use the Alphalyse silver staining protocol

Do not destain silver-stained samples!

Cut out the gel band close to the stain edges to have the highest protein concentration in the smallest gel volume. Ideal band size is 2 x 5 mm.
Do not cut the gel sample into tiny, tiny gel pieces!

Technical details

The in-gel protein sample will be reduced and alkylated with iodoacetamide, i.e. carbamidomethylated, and subsequently digested with Trypsin that cleaves after lysine and arginine residues. The resulting peptides are concentrated by Speed Vac lyophilization and redissolved before loading onto a Bruker Ancor target.

Protein identification by MALDI analysis include first a MALDI MS peptide fingerprint, followed by MS/MS sequencing of the top 15 most intense peaks in the mass range from 700 to 3000 Dalton. The data are searched against in-house protein database downloaded from UniProt containing more than 80 million known non-redundant protein sequences.

Mascot search
The Mascot software finds matching proteins in the database by their peptide masses and peptide fragment masses. The protein identification is based on a probability-scoring algorithm (www.matrixscience.com) and the significant best matching protein is shown in the Results. Homologous proteins with a lower score are not included in the report. If the protein from the correct organism is not present in the database, then a significant matching homologous protein from another organism is reported. If several proteins are identified with a significant score then several protein identifications are reported for the sample.

Results
The identified database protein sequences are shown in the Results together with the obtained mass spectrometric peptide maps. The peptides used for the identification are highlighted in the sequence and the matching peptides are listed for comparison of the determined and calculated values.The same peptide can appear as multiple identifications. It is considered a positive identification when at least 2 peptides have an Ions score above 20 or if a protein under 20 kDa has 1 peptide with an Ions score above 50. The sequence coverage is not considered for the identification. The total Mascot score provided for each identification is a total of all the individual peptide Mascot scores.

Have questions? We'd love to help.

We're always eager to help. Please send us your question or request and we will get back to you as soon as possible.

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