Protein Identification by Mass Spectrometry

This service is optimized to identify a single protein in 2D gel spots following 2D DIGE analysis. We can achieve almost 100% success rate in protein identification of 2D gel spots from our 2D DIGE projects. Please follow these guidelines in preparing and shipping samples.

For protein identification of samples containing protein mixtures such as protein extracts, IP pull downs, subcellular organelles, secretomes, cells or tissues, we offer NanoLC-MS/MS protocols optimized for each sample type.

Protein identification of 2D gel spots

Protein ID procedure: step 1 - Gel cleaning & in-gel trypsin digestion

Gel Washing

LCMSMS procedure: step 3 - Reduction/alkylation & Trypsin Digestion

Reduction / Alkylation & Trypsin Digestion

Protein ID procedure: step 2 - Desalting with C18 Zip-tip

Desalting w/ C18 Zip-tip

Abundance spectra detected by MS over the LC gradient

NanoLC-MS/MS

Protein ID procedure: step 6 - Database search using both MS and MSMS spectra to find the matching proteins

Database Search for Protein ID

Protein identification procedure:

  1. Gel treatment: 2D gel spots are washed in different buffers to remove staining dye and other inhibitory chemicals.
  2. In-gel trypsin digestion: Dried 2D gel spots are rehydrated in digestion buffer containing mass spectrometry grade Trypsin. Proteins are digested in-gel at 37°C.
  3. Peptide extraction: Digested peptides are extracted from gel with TFA extraction buffer.
  4. Desalting: The digested tryptic peptides are desalted using C-18 Zip-tips.
  5. NanoLC-MSMS optimized for 2D spots: Digested peptides are separated on a nano-flow Ultimate 3000 and analyzed by Orbitrap Exploris high-resolution mass spectrometer.
  6. Data analysis by Proteome Discoverer.
  • Protein ID Sample Report

Sample Info for 2D spots or 1D bands containing a single protein

Please keep in mind that a single band from a 1D gel could contain several proteins. This can reduce the real amount of your protein of interest. Low quantity and high complexity of samples will greatly reduce the chance of success in protein identification.

Protein amount: Our general rule of thumb is that if there is enough protein to be visible with Silver Staining or Coomassie G-250, there should be enough for Mass Spectrometry analysis. We prefer that you scale up your prep so that you have 10-20 ng of protein in the gel band. Please do not scale up by sending multiple gel pieces, as this increases gel volume and decreases the efficiency of peptide extraction from the gel. Instead, load a higher amount of protein into ONE lane of the gel.

Minimize gel volume. It is important to minimize the gel volume and maximize protein concentration. As such, we recommend you cut out only the area with your protein of interest and exclude as much unstained gel as possible.

Silver stained gel band should be compatible with Mass Spectrometry. You need to make sure your silver staining solution does not contain aldehyde that cross-links your proteins to the gel matrix, resulting in low extraction efficiency. Non-formaldehyde Silver Stain uses carbohydrazide instead of formaldehyde for reduction/development.

Shipping condition. When you are ready to send your sample, please add HPLC-grade water to cover the excised gel band in a 1.5ml eppendorf tube, or 96 well plate. Seal the Eppendorf tubes with Parafilm. Put the Eppendorf tubes into 50 mL conical tubes, then pack the 50 mL tube with Kimwipes to prevent the Eppendorfs from being crushed or jostled. The samples can be shipped on ice pack (no need to send on dry ice).

Pricing

Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
CodePriceCodePrice
LC-MS/MS on 2D Spots for DIGE Customers 1301A$229/spot301N$249/spot
LC-MS/MS on 2D Spots for External Customers 1305A$245/spot305N$265/spot
LC-MS/MS on Low Complexity Samples
(1D Gel band, 3-5 samples) 1		331A$499/sample331N$599/sample
LC-MS/MS on Low Complexity Samples
(1D Gel band, 6 or more samples) 2		331A$459/sample331N$539/sample
LC-MS/MS on Moderate Complexity Samples 2332Acall for price332Ncall for price
LC-MS/MS on Moderate to High Complexity Samples 2334Acall for price334Ncall for price
LC-MS/MS on High Complexity Samples 2333Acall for price333Ncall for price

For those customers who used our upstream 2D DIGE services, we offer a significant discount on the protein ID cost so they can complete their project at a low cost. This is why the cost of protein ID is much lower for DIGE customers than the external ones.

1. Each order should have a minimum of 5 samples. Turnaround time is ~5 business days. Price covers the following:

  • Gel treatment
  • In-gel trypsin digestion
  • Peptide extraction
  • NanoLC-MS/MS with optimized gradient for 2D spots
  • Standard database search against one species in NCBI or SwissProt database
  • Protein ID Sample Report

2. Each order should have a minimum of 2 samples. Turnaround time is 5-7 business days for 3 or more samples, and up to 10 business days for 2 samples.

Sample types:

  • Low complexity samples: 1D gel band – Each sample should contain only one gel piece with one protein band, no bigger than 1x1x5mm in size, <10 proteins. Samples with more than one gel piece, and/or more than one protein band will be considered as moderate to high complex samples. We recommend you cut out only the area with your protein of interest (1x1x2mm size) and follow these guidelines to prepare samples
  • Moderate complexity samples: protein mixture such as column purified sample, <100 proteins 
  • Moderate to High complexity samples: Protein mixtures such as subcellular organelles, IP samples
  • High complexity samples: whole cell lysate, tissue, and others

Price covers the following:

  • Nano-LC coupled with auto-spotting
  • MS/MS
  • Standard database search against one species in NCBI or SwissProt database using MASCOT

Price does NOT cover:

  • Sample preparation
  • Data request in addition to the standard report