Peptide mapping

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

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.

We digest your sample using 1-5 enzymes of choice. Each of the resulting peptide mixtures is analyzed by LC-MS/MS. Then the observed peptides are mapped to the theoretical amino acid sequence of your drug substance protein.

Service includes:

  • Your sample is digested with 1-5 enzymes, such as trypsin, Glu-C, and chymotrypsin.
  • Mass spectrometry analysis is done by nano LC-MS/MS or UV LC-MS/MS.
  • We perform peptide mapping with comparison to your expected amino acid sequence.
  • You receive a sequence coverage map.
  • You receive a list of all observed peptides.

Optional:

  • Digestion of your sample with more proteases (trypsin, Lys-C, Glu-C, chymotrypsin, Asp-N, Arg-C…).
  • Your choice of LC gradient and column.
  • Your choice of MS instrument (MALDI MS/MS, QTOF, TrippleTOF).
  • 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 a request using our request form. 

Order now

Peptide mapping with 2 proteases and nanoLC MS/MS

• High sequence coverage of your protein
• Digest with 2 enzymes; trypsin, chymotrypsin
• Sensitive nanoLC MS/MS QTOF analysis
• Common modifications considered (deamidation, Met/Trp oxidation)
• Combined sequence coverage map is typically 70-95%

$1,750.00

World Class Service
Quality analysis, presented in easy-to-understand reports

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
• Sequence coverage generally 75-85%

$1,750.00

World Class Service
Quality analysis, presented in easy-to-understand reports

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

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

When you go through our online ordering process you will have the possibility to chose from 4 different shipping methods.

1. Ship samples using courier company of your choice

Place your samples into micro tubes and pack in a suitable transport box

  • Contact a courier company of your own 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
  • For shipping of samples to be stored below 5°C/40°F use cool packs or cooling units
  • Ship the samples to the office nearest to you:
Customers in North America

Alphalyse, Inc.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA

Att: Jette Williams
Tel: (650) 543-3193
Fax: (650) 230-1251 E-mail: info@alphalyse.com

Customers in Europe

Alphalyse A/S
Unsbjergvej 4
DK-5220, Odense
DENMARK

Att: Anette Rasmussen
Tel: +45 6310 6500
Fax: +45 6310 6509 E-mail: info@alphalyse.com

 

2. Ship samples with FedEx overnight, prepaid (room temperature)

Prepaid FedEx Air Waybills as PDF can be used for shipment of samples for packages up to 1 lb./ 500 g, stored at room temperature. . An Air Waybill is a prepaid shipment paper needed for national and international shipments. If you select this shipping type during the order process, we will generate an prepaid Air Waybill and send it to you by e-mail (PDF-format) within 24 hours.

Place your samples into micro tubes. Pack in suitable transport box

  • Print the PDF and stick it on the transport box
  • Follow the simple instructions on how to request a pickup from your local FedEx office.

 

3. Ship samples with FedEx overnight, prepaid (below 5°C/40°F)

Prepaid FedEx Air Waybills as PDF can be used for shipment of samples for packages up to 10 lb./ 5 kgs, with cooling packs. An Air Waybill is a prepaid shipment paper needed for national and international shipments. If you select this shipping type during the order process, we will generate an prepaid Air Waybill and send it to you by e-mail (PDF-format) within 24 hours.

Place your samples into micro tubes. Pack in suitable transport box. Fill with cooling units/cold packs.

  • Print the PDF and stick it on the transport box
  • Follow the simple instructions on how to request a pickup from your local FedEx office.

 

4. Ship samples with World Courier, prepaid

The Prepaid 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.

  • 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