Home > Proteomics Services > Mass Spectrometry Service
Our Mass Spectrometry services offer almost 100% Success Rate in protein identification, by employing the lastest technologies and optimized protocols. Customers can choose from the following:
A. MALDI TOF/TOF: Identify a single protein in 2D spots or 1D gel bands
B. Nano LC-MS/MS: Identify ALL proteins in a mixture ranging from 1D gel bands, subcellular organelles, IP samples, cell culture media to whole cell lysate or tissues
A. MALDI TOF/TOF Procedures (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 assay performance.
Reduction, Alkylation, Gel Washing & In-gel Trypsin Digestion |
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Desalting w/ Zip-tip C18 (Millipore) |
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Spotting on MALDI plate |
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MALDI-TOF |
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MALDI-TOF/TOF |
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Database Search for Protein ID |
1. Gel treatment: 2D gel spots
are washed multiple times to remove
staining dye and other inhibitory
chemicals. Gel spots are then dried to
absorb maximum volume of digestion
buffer.
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 and shaking.
4. Desalting: The digested
tryptic peptides are desalted using C-18
Zip-tips (Millipore).
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 using an Applied Biosystems
Proteomics Analyzer.
7. MALDI-TOF/TOF: Ten to twenty
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 GPS Explorer
software equipped with the MASCOT search
engine to identify proteins from primary
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 a sample report of protein ID analysis)
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Guidelines for Preparing and Shipping Samples for Protein Identification
1-D gel band: Please keep in mind that a band from a 1D gel could contain several proteins, although it looks a single band. 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 for high confident identification. Please follow the tips below:
1. 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.
2. 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 (1x1x2mm size) and exclude as much unstained gel as possible.
3. 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.
4. 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) to the following address:
Proteomics Service
Applied Biomics, Inc.
23785 Cabot Blvd, Suite 312
Hayward, CA 94545
USA
If you are interested, please follow these steps to obtain more information and order this service.