face

Identify single proteins by MALDI-MS

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

We offer single protein identification of your 1D gel band or 2D gel spot with an efficient MALDI-MS analysis.

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.

We search the obtained data against a public protein database from UniProt. This database contain amino acid sequences for more than 100 million proteins. We update the databases each month to contain all known proteins. Trough automation, the analysis have a throughput of more than 100 samples per day.

The service includes:

  • Send the gel band/spot for analysis and we perform in-gel reduction/alkylation and trypsin digest.
  • After digest, we run of MALDI MS and MS/MS analysis.
  • This is followed by a MASCOT search in NCBI/UniProt protein database.
  • Finally, we can identify single proteins for each sample.
  • You thus receive the analysis report within 4-8 days.

Please send us a request with a description of your project, so we can tailor your project proposal. 

Services
Identification of single or multiple proteins

Protein Identification

  • 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

200.00

Fast and Easy
Will be delivered in 4-8 days
Info / Order
Request quote

First we reduce the in-gel protein sample and alkylate it with iodoacetamide, i.e. carbamidomethylated. Subsequently a digestion with Trypsin cleaves after the lysine and arginine residues. Speed Vac lyophilization then concentrates the resulting peptides, which we redissolve before loading onto a Bruker Ancor target.

Protein identification by MALDI analysis includes first a MALDI MS peptide fingerprint. MS/MS sequences the top 15 most intense peaks in the mass range from 700 to 3000 Dalton. Finally, we search the data an in-house protein database downloaded from UniProt. This database contains 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. It bases the protein identification on a probability-scoring algorithm (www.matrixscience.com) and shows the significant best matching protein in the Results. We leave homologous proteins with a lower score out of the report. If no protein from the correct organism is present in the database, it then reports a significant matching homologous protein from another organism. If the software identifies several proteins  with a significant score it then reports several protein identifications for the sample.

Protein ID – 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.

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!

shipping
Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box. Remember to print and include the sample submission form.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
Customers in the USA
Alphalyse, Inc.
299 California Avenue, Ste 200
Palo Alto, CA 94306
USA
Att: Thomas Kofoed
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220 Odense SO
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
shipping
Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack in suitable transport box. Remember to print and include the sample submission form.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.

shipping
Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Remember to print and include the sample submission form.
  • Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

shipping
Get a quote for shipment by World Courier

The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork
Order analysis now
Sign up for the newsletter
Subscribe to get 3 tips that will instantly improve your protein analysis results

Receive monthly emails with news, tips, case stories
and offers for protein analysis
Private, secure, spam-free
© Copyright Alphalyse 2002 - 2019. All rights reserved.