Protein Identification by MALDI-TOFTOF

Protein Identification by MALDI-TOFTOF is mainly used 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. In each run, four sensitivity standards in the amount of 1 femtomole are used to monitor the performance.

Protein identification by MALDI TOF/TOF procedure

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

Gel Washing & In-gel Trypsin Digestion

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

Desalting w/ C18 Zip-tip

Protein ID procedure: step 3 - Spotting on MALDI plate

Spotting on MALDI plate

Protein ID procedure: step 4 - peptide finger printing by MS


Protein ID procedure: step 5 - peptide fragmentation by MSMS


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 sequencing grade modified 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. Spotting: The desalted peptides are mixed with CHCA matrix (alpha-cyano-4-hydroxycinnamic acid) and spotted into wells of a MALDI plate.
  6. MALDI-TOF: Mass spectra (MS) of the peptides in each sample are obtained.
  7. MALDI-TOF/TOF: 10-20 of the most abundant peptides in each sample are further subjected to fragmentation and tandem mass spectrometry (MS/MS) analysis.
  8. Database search: Protein identification is based on peptide fingerprint mass mapping (using MS spectra) and peptide fragmentation mapping (using MS/MS spectra). Combined MS and MS/MS spectra are submitted for database search using the MASCOT search engine to identify proteins from NCBI or SwissProt sequence databases.
  9. Data Report: Includes the top 10 highest scoring hits from the database search for each 2D gel spot, as well as a summary listing the best match for each sample (see an example below)

Sample Info for gel bands (1D) or spots (2D)

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 by MALDI-TOF/TOF. In general, MALDI-TOF/TOF is ideal for identifying a single protein from 2D gel spots. Please follow the tips below:

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).


Services DescriptionAcademic/Governmental LabsIndustrial/Commercial Labs
Protein ID by MALDI-TOF/TOF for DIGE Customers 1104A$139/sample104N$159/sample
Protein ID by MALDI-TOF/TOF for External Customers
(3-10 samples) 1
Protein ID by MALDI-TOF/TOF for External Customers
(more than 10 samples) 1
LC-MS/MS on Low Complexity Samples
(1D Gel band, 2-5 samples) 2
LC-MS/MS on Low Complexity Samples
(1D Gel band, 6 or more samples) 2
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 3 samples. Turnaround time is 2-5 business days. Price covers the following:

  • Gel treatment
  • In-gel trypsin digestion
  • Peptide extraction
  • Desalting
  • Spotting
  • Standard database search against one species in NCBI or SwissProt database using MASCOT

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