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Peptide mapping

Confirmation of theoretical amino acid sequence by LC-MS/MS peptide mapping

Are you seeking to confirm the amino acid sequence of your purified protein by peptide mapping?

Peptide mapping by LC-MS/MS is one of the most valuable methods for verifying the amino acid sequence of your drug substance protein. Does your company follow the ICH Q6B Guidelines? According to ICH Q6B you must then provide peptide mapping to confirm the amino acid sequence.

The service includes:

  • First we digest your sample with 1-5 enzymes, such as trypsin, Glu-C, and chymotrypsin.
  • Then mass spectrometry analysis is made by nano LC-MS/MS or UV LC-MS/MS.
  • We finally perform peptide mapping with comparison to your expected amino acid sequence.
  • You receive a sequence coverage map.
  • We also include a list of all observed peptides.

Optional:

  • Digestion of your sample with more proteases (e.g. trypsin, Lys-C, Glu-C, chymotrypsin, Asp-N, Arg-C).
  • Your choice of LC gradient and column.
  • Your choice of MS instrument (choose among MALDI MS/MS, QTOF, TrippleTOF).
  • A search for specific postranslational modifications and amino acid substitutions in your sample.

If none of the above meets your requrements, you are welcome to send us a request

Looking for simple peptide mapping for confirmation of theoretical amino acid sequence – by MALDI-MS/MS or nano LC-MS/MS? Then take a look at our PICK ‘n POST services.

Services/Products
Protein analysis by Alphalyse

Peptide mapping with 2 proteases and nanoLC MS/MS

  • Digest with 2 enzymes; trypsin, chymotrypsin
  • Sensitive nanoLC MS/MS QTOF analysis
  • Common modifications considered (deamidation, Met/Trp oxidation)

$1,750.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
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Protein analysis by Alphalyse

Peptide mapping of drug substance by UV LC-MS/MS

  • Digestion with 1 enzyme, eg. trypsin, Lys-C
  • UV-LC-MS/MS, 60 min gradient
  • Common modifications included, eg deamidation, oxidation

$1,750.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote
Protein analysis by Alphalyse

Peptide mapping of drug substance, customized

  • Peptide map by UV-LC MS, nLC-MS, or MALDI MS
  • Choice of 1-5 proteolytic enzymes
  • Custom LC MS conditions; gradient, column
  • Search for specific modifications and amino acids

$3,000.00

World Class Service
Quality analysis, presented in easy-to-understand reports
Info / Order
Request quote

Introduction
In Mass spectrometric Peptide Mapping the protein is cleaved into smaller peptides using a specific protease. The peptides are analyzed by mass spectrometry and the observed peptides correlated to the protein amino acid sequence.

The identified peptides in the peptide map thus confirms the specific amino acid sequences covered by the peptide map, as well as the identity of the protein. Peptide mapping using a single protease will typically give a sequence coverage of 30-90%. By using two or more proteases this coverage can be increased to 95-100%.

Detailed characterization of a protein requires careful investigation of the protein sequence to select the best proteases, and choice of nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides. The difference between the two techniques is related to the amount of material needed for the analysis – nano-flow can be conducted on low microgram protein whereas the high-flow analysis requires at least 20 microgram per analysis. The nigh-flow analysis include a UV-trace of the chromatogram.

nano-flow LC-MS/MS
The peptide mapping using nanoLC-MS/MS is performed on a Dionex 3000 system running at 300 nl/min equipped with C18 capilary column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provide a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge from 200-1600 dalton.

high-flow LC-MS/MS with UV
The peptide mapping using high-flow LC-MS/MS is performed on a Agilent 1200 system running at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument is running either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provides both a UV- and a Base peak chromatogram, and all MS/MS data is collected for peptides with mass/charge from 200-1600 dalton.

Introduction
Protein identification by mass spectrometry requires a pure protein in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

Protein samples can be submitted in liquid or lyophilized.

  1. Purify the protein/peptide

    1. The chromatographic protein/peptide purity should be >90%
    2. 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
    3. Minimum amount ~ 10-50 micrograms
  2. Put the protein sample into microcentrifuge tube

    1. Use a Eppendorf Safe Lock tube supplied, or similar tubes from your lab
    2. Lyophilize or speed vac the protein to a solid sample to ensure protein stability during shipment
    3. Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample

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
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.

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. 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!

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
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