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Peptide mapping against sequence

Amino acid sequence confirmation by peptide mapping

Peptide mapping for confirmation of theoretical amino acid sequence – by MALDI-MS/MS or nano LC-MS/MS 

Are you looking for a service to map your peptides and confirm them to a theoretical aa sequence?

We digest your protein using Trypsin. This cleaves the sequence after Arg and Lys residues. The resulting peptide mixture is then :analyzed by either MALDI-MS/MS or nano LC-MS/MS. The observed peptides are mapped to the expected amino acid sequence.

Service includes:

  • We do in-gel trypsin digest and peptide mapping by MALDI-MS/MS or nano LC-MS/MS.
  • We compare with your theoretical amino acid sequence.
  • You obtain a sequence coverage map.
  • You obtain calculated sequence coverage in % (generally 20-100%).
  • Report is delivered within 4-8 days.

How-to-order-PICK-n-POST

Do you have further needs?

– send us a request. We would like to enter into dialogue with you to meet your specific needs.

Examples from report:

Services/Products
Peptide sequence by Nano LC-MS/MS

PICK ‘n POST™ peptide mapping by MALDI-MS/MS

  • Reduction/alkylation of cysteines
  • Protein in-gel trypsin digest
  • MALDI-MS/MS peptide analysis
  • Peptide mapping against custom aa sequence

$550.00

Fast and Easy
Will be delivered in 4-8 days
Info / Order
Request quote
Peptide sequence by Nano LC-MS/MS

PICK ‘n POST™ peptide mapping by nano LC-MS/MS

  • Reduction/alkylation of cysteines
  • Protein in-gel trypsin digest
  • nanoLC-MS/MS peptide analysis
  • Peptide mapping against custom aa sequence

$800.00

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

Technical details

Introduction
Peptide mapping is a vital tool in the process of confirming the expected amino acid sequence of your protein.

It is used for in-depth protein analysis that can identify minor differences in the primary structure such as errors in the transcription, point mutations, and post-translational modifications.

There are many pitfalls in peptide mapping including incomplete digestion, non-reproducible digestion conditions, non-specific cleavages and non-reproducible chromatography. We at Alphalyse have years of experience in selecting the best digest conditions, ensure enzyme activity and specificity, and performing the mass spec analysis with robust and reproducible protocols. We always include standards that has to fulfill preset criteria to approve your analysis results.

The sequence coverage using a single in-gel digest will typically be between 20 and 100% dependent on the protein amount and the specific protein sequence. Our standard PICK ‘n POST™ analysis is carried out by digesting your in-gel protein sample using Trypsin which cleave the amino acid sequence after Arg and Lys residues . The resulting peptide mixture is analyzed by either MALDI MS/MS or nano LC-MS/MS, and observed peptides are mapped to the expected amino acid sequence. We provide you with a peptide coverage map, illustrating exactly which amino acid sequences that were identified. We also calculate the sequence coverage in percentage. The sequence coverage is typically higher for nanoLC-MS/MS analysis compared to MALDI-MS/MS analysis.

Peptide mapping with MALDI-MS/MS
Peptide mapping with 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 combined data is searched against the custom sequence, with standard settings (variable modification Met oxidation).

Peptide mapping with nanoLC-MS/MS
Peptide mapping with nanoLC-MS/MS include a 30 minute gradient on C18 Reverse Phase  nanoLC system connected to a high resolution Q-Tof instrument. The Q-Tof instrument is running in data dependent acquisitions mode (DDA), in which a fixed number of precursor ions recorded in a survey scan are selected to a second stage of MS/MS fragmentation analysis. All MS/MS data is combined in a database search against custom sequence.

Sample requirements

Introduction
Peptide mapping 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.

Samples for our PICK ‘n POST peptide mapping can only be delivered as In-gel samples

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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