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Identify multiple proteins – nanoLC-MS

Identification of protein mixtures in-gel bands or in-solution by nanoLC MS/MS

Do you have several proteins present in a gel band excised from 1D SDS PAGE? We can help you identify these proteins. Just send the gel band – Then we perform the analysis by trypsin digestion followed by nanoLC-MS-MS peptide analysis and database searching.

We search the data against the public NCBI and UniProt databases. They contain the amino acid sequence of more than 80 million known proteins. We update the databases on a monthly basis to ensure access to 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.

In-gel samples

ID of multiple proteins by nano LC-MS/MS (in-gel)

  • Trypsin cleavage and nanoLC-MS/MS peptide analysis
  • Search against protein sequence database
  • ID of up to 10-40 proteins
$550.00
Per sample
Results in 4-8 days
In-solution samples

ID of multiple proteins – nano LC-MS/MS (in-solution)

  • Trypsin cleavage and nanoLC-MS/MS peptide analysis
  • Search against protein sequence database
  • ID of up to 10-100 proteins
$800.00
Per sample
Results in 10-20 days
Service includes
  • You provide us with gel band containing multiple proteins.
  • We perform in-gel trypsin digest and nanoLC-MS-MS peptide analysis.
  • We search data against protein sequence databases for protein identity.
  • You obtain identification of typically 10-40 proteins per sample.
  • The report is delivered within 4-8 working days.

 

difference between 1d and 2d gel electrophoresis

Three different ways for preparing complex In-gel samples for protein identification

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

Indentification of several proteins present in a gel band excised from 1D SDS PAGE

Sample requirements

Short 1D PAGE: The protein sample is loaded onto a 1D PAGE gel and run for only 1 cm (5-8 min at 200 V). After staining with Coomassie, the entire stained area is excised as one sample and sent for analysis.

Long 1D PAGE: The protein sample is loaded onto a 1D PAGE gel and run as normal. After staining with Coomassie, the entire stained area is excised into 5-10 smaller pieces. Place each piece in separate tubes and ship these for analysis.

2D PAGE: The protein sample is loaded onto a 2D PAGE gel and run as normal. After staining with Coomassie, selected areas with multiple spots are excised. Place each piece in separate tubes and ship these for analysis.

Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS can be done on samples containing small amounts of salts, urea or detergent, but the best result are obtained with low buffer strength in volatile solvents without detergents

Technical details

The protein samples 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 for injection on a Dionex nano-LC system and MS-MS analysis on a Bruker Maxis Impact Q-TOF instrument. The MS-MS spectra are used for Mascot database searching. The data are searched against in-house protein database downloaded from UniProt containing all known non-redundant protein sequences.

Database 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.

Reporting
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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