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Proteomic Services

Refer to Protein Sample Submission and Guidelines and Protocols for recommendations and information prior to preparing and submitting protein gel bands or protein samples for analysis.

Proteomics Service What is Performed at the Facility
Protein ID from SDS Gel
Identification of proteins from 1D gel bands or 2D gel spots

Digests available with trypsin or chymotrypsin only
  1. Excise gel bands or spots
  2. In-gel protein digest
  3. nanoLC/MS/MS analysis
  4. Mascot database search and report of results
Protein ID from Solution
Identification of proteins from affinity purifications, HPLC, or other purified protein solution

Digests with alternative enzymes are available (e.g. trypsin, chymotrypsin, pepsin, thermolysin, Asp-N, Glu-C)
  1. Ziptip purification, if required
  2. In-solution protein digest
  3. nanoLC/MS/MS analysis
  4. Mascot database search and report of results
Protein ID from Complex Isolates in Solution and IP Pull-down Experiments
Identification of proteins from affinity purifications, cell extracts, tissues, and other complex mixtures in solution

SCX fractionation of peptides required with complex mixtures prior to nanoLC/MS/MS (i.e. “Mudpit”-type analysis)

Digests with alternative enzymes are available (e.g. trypsin, chymotrypsin, pepsin, thermolysin, Asp-N, Glu-C)
  1. Ziptip purification, if required
  2. In-solution protein digest
  3. SCX fractionation, if required
  4. nanoLC/MS/MS analysis of fractions
  5. Mascot database search and report of results
Identification of Protein Modifications Post-translational Modifications
Mapping of post-translational modifications or derivatives. (e.g. phosphorylation, ubiquitination, acetylation)

Specific modifications of interest can be targeted during nanoLC/MS/MS data acquisition.

Digests with alternative enzymes are available (e.g. trypsin, chymotrypsin, pepsin, thermolysin, Asp-N, Glu-C)

IMAC isolation of phosphopeptides is available TiO2 isolation of phosphopeptides (available soon)

When possible, ESI/MS analysis of intact proteins can be invaluable for confirmation of protein sequence or identification of truncated sequence or post-translational modifications.
 Often, identification of protein modifications requires several mass spec experiments and may involve different strategies : (discuss options with Facility personnel)

Analysis Options:
  1. Modifications can be identified through analysis of protein digests and database searches.
      Protein digests can be analyzed by nanoLC/MS/MS, Maldi/ToF, Maldi/FTMS
  2. Modifications can be found through accurate MW measurement of intact proteins.
      Intact proteins analyzed by ESI/MS Molecular mass spectrum is generated Protein sequence is confirmed, or other truncation or sequence variant found, or post-translational modifications indicated
  3. Modifications can potentially be located by Top-down analysis of intact proteins with ECD fragmentation and FTMS of the intact molecule
Intact Protein MW Analysis
ESI/MS analysis and deconvolution of data using MaxEnt software
(Xtract or ProMass software with FTMS analysis)
  1. Ziptip purification, usually required
  2. ESI/MS analysis
  3. MaxEnt or ProMass deconvolution of data into a neutral molecular-mass spectrum
  4. Search protein sequence for truncation, cut sites, degradation products, or sequence modification with Biolynx software (if requested)
Peptide Sequencing with MS/MS (FTMS)
and accurate mass denovo Sequencing
CID, ECD, or IRMPD fragmentation methods available with FTMS analysis of the product ions Sample introduced by infusion ESI or nanospray ESI (pico-tip); Samples can also be ionized using AP-MALDI
  1. Ziptip purification (usually required)
  2. ESI/MS/MS or Maldi/MS/MS sequencing of peptides)
  3. Mascot or PEAKS denovo sequence search and prediction scores and results
Disulfide Linkage Characterization
Determine protein disulfide linkages

May require multiple experiments and analyses: for example, digests with trypsin, chymotrypsin, pepsin; derivatization with NEM, MMTS;
LC/MS/MS (FTMS) and/or Maldi/MS/MS (FTMS)
 Identification and mapping of protein disulfide linkages may require several mass spec experiments involving different strategies: (discuss options with Facility personnel) Analysis Options:
  1. Intact protein MW measurement by ESI/MS if feasible to determine the number of linkages present
  2. Reduce the protein with DTT, determine intact MW shift and confirm the number of linkages present
  3. Derivatize the protein with NEM or MMTS Measure the intact MW and confirm the number of blocking groups (or free sulfhydryls)
  4. Digest with chymotrypsin LC/MS/MS analysis Maldi/MS/MS analysis
  5. Data search for linked peptides
Identification of Sulfur-containing peptides
(i.e. containing cysteine or methionine )
FTMS with high resolution separation of heavy isotopes, (e.g. 13C and 34S isotopes; limited to peptides < 1000 MW)
  1. Ziptip purification, if required
  2. ESI/MS (high resolution FTMS) of 2nd or 3rd isotope peaks of peptides
  3. Theoretical software calculations of isotope patterns
  4. Compare experimental and theoretical isotope patterns to establish presence of sulfur.
“Top-Down” Proteomics
FTMS with ECD fragmentation and sequencing intact proteins and peptides
Inquire
Protein Expression/ Quantitation Analysis
ICAT, ITRAQ experiments
Peptide quantitation
 Inquire


Custom Database Searching:
Create a custom Mascot database
For specific protein sequences not in the MSDB or NCBI databases (i.e. engineered protein sequences, variants in known protein sequence, putative protein sequences)
  1. Create a custom database in Mascot with your specific protein sequences to be used for protein ID or modification searches
Create a custom modification for Mascot searches
For searching custom or non-standard modifications or derivatives
  1. Create a custom modification option in Mascot for searching your protein data
Special request/ additional modification searches with Mascot or additional database searches
Other database search engines available on request (e.g. Sequest, PEAKS , Protein Prospector , OMSSA)

Each search includes up to three modifications and report of results
  1. Database searches with Mascot using your specific protein sequence or modification requests
  2. Database searches using other database search engines (e.g. Sequest, PEAKS , Protein Prospector , OMSSA).
  3. Report of search results

Enzyme Digestion of Protein Samples

In-gel Protein Digest*

1D gel bands or 2D gel spots are preferably excised and destained by personnel at the MS Facility. In-gel digests can be performed with trypsin or chymotrypsin enzymes only.

The following gel stains are acceptable. However, coomassie, Colloidal Blue (Invitrogen) and other blue-type stains are preferred for protein ID from in-gel analysis.
  • Coomassie
  • Colloidal Blue
  • Silver (i.e. mass spec compatible, see special submission requirements)
  • Sypro-ruby
(See Sample Submission and Guidelines and Protocols for recommendations prior to preparing, running, and staining gels).

* We do not offer gel electrophoresis or gel staining services; the user must provide the gel or gel band/spot to the MS Facility.

Protein Solution Digest

Digests of proteins in solution can be performed with the following enzymes. However, trypsin and chymotrypsin are preferred and will be the most specific and informative in most cases.

  • Trypsin
  • Chymotrypsin
  • Thermolysin
  • Pepsin
    For other proteases (e.g. Asp-N, Glu-C, O-glycosylase, N-glycosylase) or other reagents, such as CNBr, please inquire.

(See Sample Submission and Guidelines and Protocols for recommendations prior to preparing protein samples in solution).

Identification of Protein Post-translational Modifications and Derivatives

Identification and mapping of post-translational modifications can be difficult, particularly when the modification is at relatively very low levels. Often, several mass spectrometry experiments may be required and may involve different sample preparations and mass spectrometry strategies.

For example, if the type and extent of modifications are unknown, a combination of analysis strategies are needed. When feasible, ESI/MS analysis of the intact protein can be highly informative in determining whether the protein has any modifications, what those modifications may be (such as phosphorylation), at what approximate levels, and whether the protein amino-acid sequence is different than anticipated, truncated, or degraded from protease activity.

Analysis of protein digests is used to identify and map sites of modification. When searching for possible modifications, it is recommended analyzing the protein using two or more different enzyme-digest experiments, with subsequent nanoLC/MS/MS analyses and/or Maldi/MS/MS analyses, and multiple database searches for possible modifications. Accurate mass measurements with FTMS (LTQ-FT instrument), along with MS/MS sequence, provide a high-degree of confidence in the assignment and identification of modified peptides.

Phosphorylations can be targeted using IMAC (immobilized metal affinity chromatography) or TiO2 columns to selectively isolate and purify phosphopeptides from the digest. These procedures can sometimes greatly improve the ability to find phosphorylations.

If a specific site or region of the protein is anticipated to be modified (such as phosphorylation), a custom method for the mass spectrometry analysis can be setup (i.e. create an inclusion list of ions) to specifically target one or more putative modified peptides of interest, thereby increasing the capability of the instrument to identify those peptides or modified peptides.

Proteins with labile modifications (such as GlcNAc or phosphorylations) can be identified using ECD (electron-capture dissociation) fragmentation of peptides for MS/MS sequencing. The ECD technique (in the FTMS instrument) generally leaves labile modifications intact during fragmentation of peptides, such that the position of the modification can be clearly determined. Other lower-energy fragmentation methods, such as CID (collision-induced dissociation) or IRMPD (infrared multiphoton dissociation) can result in dissociation of labile modifications, but often the modification and site of modification can still be identified by virtue of mass-loss differences observed in the MS/MS sequence spectra. Modifications can potentially be located by “top-down” analysis of intact proteins using ECD fragmentation of the intact protein in the FTMS.

Database searches using Mascot software and other search engines are used to process the MS/MS spectra of peptides to locate the sites of modifications.

Many types of protein modifications can potentially be characterized, such as:

(See Sample Submission and Guidelines and Protocols for recommendations and information prior to preparing protein samples for analysis of modifications)

Use the links below to learn about specific services:

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