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Identify single proteins by MALDI MS

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

We offer PICK ‘n POST™ identification of single protein by MALDI MS-MS analysis

Would you like to identify the single most abundant protein present in a 1D gel band or 2D gel spot? We can help you identify this protein by its mass spectrometric peptide fingerprint. Just send us the gel band/spot. Then we perform trypsin in-gel digestion. Which 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 NCBI or UniProt. These databases contain aa sequences for more than 80 million proteins. Furthermore, each month the databases are updated to consider all known proteins.

Our service includes:

  • You provide us with gel band/spot for analysis.
  • We then 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.

How-to-order-PICK-n-POST

Do you want to analyze multiple proteins present?

No problem! We can identify multiple proteins by nanoLC-MS-MS.

If you have further needs

  • If you want us to run the 1D/2D gel.
  • Or you want identification of several proteins in each gel band/spot.
  • Perhaps you have specific protein sequences you want us to include in the database search.
  • Maybe you need a high throughput identification of many samples.

Simply send us a request, so we can enter into dialogue with you and meet your specific needs.

Examples from report

 

Services/Products
Identification of single or multiple proteins

PICK ‘n POST™ identification of single protein by MALDI-MS/MS

  • You provide gel band/spot
  • We perform peptide analysis by MALDI-MS/MS
  • Search against UNIPROT database
  • Provide identification of single protein in sample
  • Sequence coverage map for the identified protein

$225.00

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

Technical details

Introduction

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 databases downloaded from UniProt and NCBI 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 35 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.

Sample requirements

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

 

In-gel samples:
Proteins from 1D SDS PAGE and 2D PAGE gels can be submitted for peptide mapping.
Run your 1D/2D gel
Avoid fingerprints – wear gloves, without talcum powder
Use clean glassware and staining trays
All gel handling should take place in a dust free lab. Dust contains human keratin!

Stain the gel with Coomassie Blue, SYPRO Ruby or Silver stain
Coomassie Blue and SYPRO Ruby works well in combination with mass spectrometry
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 bands/spots of interest
Use a clean scalpel and tweezers
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!
Clean the scalpel in-between spots/bands

Put the samples into separate wells/tubes with water
Add 100-200 μl distilled water to keep each gel plug wet.

Sample shipment

shipping option
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.

  • 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.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
shipping option
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.
  • 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 option
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.
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. Fill with cooling units/cold packs.
  • 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.

shipping option
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

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