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Identify multiple proteins by nanoLC-MS

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

We offer PICK ‘n POST™ identification of multiple proteins by nanoLC-MS-MS

Do you have several proteins present in a gel band excised from 1D SDS PAGE or in a gel spot excised from 2D PAGE or in-solution? We can help you identify these proteins. Just send us the gel band/spot/in-solution sample. – Then we perform the analysis by trypsin digestion followed by nanoLC-MS-MS peptide analysis and database searching.

We search your data against 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.

Service includes:

  • You provide us with samples containing multiple proteins.
  • We will then perform in-gel trypsin digest or in-solution 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.
  • A report is delivered to you within 4-8 working days.

How-to-order-PICK-n-POST

Do you have further needs?

  • You want us to run the 1D/2D gels and cut the bands/spots for the identification analysis.
  • Need identification of more than 20 proteins in each gel band/spot.
  • Looking for a high sequence coverage map for each identified protein.
  • You have specific protein sequences you want included in the database search.

– then send us a request, so we can design an analysis to your specific project.

Examples from report

Services/Products
Identification of single or multiple proteins

PICK ‘n POST™ identification of multiple proteins by nano LC-MS/MS in-gel samples

  • You send gel band with multiple proteins
  • Trypsin cleavage and nanoLC-MS/MS peptide analysis
  • Search against protein sequence database
  • Identification of up to 10-40 proteins
  • Report delivered within 4-8 days

$550.00

Fast and Easy
Will be delivered in 4-8 days
Info / Order
Request quote
Identification of single or multiple proteins

PICK ‘n POST™ identification of multiple proteins by nano LC-MS/MS in-solution

  • You send sample in-solution with multiple proteins
  • Trypsin cleavage and nanoLC-MS/MS peptide analysis
  • Search against protein sequence database
  • Identification of up to 10-100 proteins
  • Report delivered within 4-8 days

$800.00

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

Technical details

Introduction
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 databases downloaded from UniProt and NCBI containing more than 80 million 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 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

Sample preparation

You can prepare the gel bands/spots for this analysis in three ways:

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.

Cut out the bands/spots of interest
Use a clean scalpel and tweezers
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.

For in-solution samples please follow these instructions:

In-solution samples: The p

Protein samples can be submitted in solution or lyophilized. Minimum amount ~ 10-50 micrograms

 

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

Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab

Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment

Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample

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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