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Mass spectrometric proteomics

Core Facility service:

Identification of proteins

The service of the Core Facility comprises identification of proteins in SDS-PAGE-bands and 2DE-Gel-spots.

For analysis of proteomes, special forms of protein analysis or special mass spectrometric analysis (all of these forms of analysis do not belong to the Core Facility service but are collaborations), please contact us at MS@uke.de or at Phone: 040-7410-58795 or -51907.

In SDS-PAGE-bands / 2DE gel spots

Instruction for getting a protein analysis of a SDS-PAGE band / 2DE gel spot:

CAUTION 1: Please read and follow our sample preparation guidelines for gel preparation, otherwise successful results cannot be guaranteed due to contamination with e.g. by keratin.

CAUTION 2: The silver staining kit must be mass spectrometry compatible. Please use the silver staining kit from Proteom Factory (FireSilver).

Complete the MS-request form, available .

PLEASE NOTE: We are not allowed to process your samples, if your third-party funding account number is missing on the request.

      Guidelines: Print the MS-request form. Get the signature of the owner of the third-party fund account (Drittmittel-Konto-Inhaber) or the order oblige (CS-Bestellberechtigter). Cut the stated label describing unambiguously your sample from the request form. Fix the label with sticky tape (e.g. Tesa-Film) on the corresponding tube.Place the MS-request form together with the sample tubes into an envelope. Put the envelope into our CF-MP Mailbox (Located next to our office N27, ground floor, room 00.008)      

In electrophoresis gel bands or 2D electrophoresis spots

Protein identification via mass spectrometric analysis of tryptic peptides, generated by proteolytic digestion (by trypsin) of the protein(s) in the SDS-PAGE band or 2DE gel spots.

 

 Protein identification by the search engine Mascot

 ®MS/MS-data converted into a peaklist file (e.g. mgf or mzML), which we can send upon request.

 ®Using the peaklist file you can perform protein identification by yourself with the search engine Mascot (http://www.matrixscience.com/)

 ®For using Mascot please read the Mascot-SOP for protein identification (Analysis via LC/ESI-Q-TOF or LC/ESI-IT-MS).

Support in protein analysis (performed in collaboration projects)

Consulting with respect to integration of proteomics in answering scientific question

Molecular weight determination of proteins and peptides

Molecular weight determination of intact peptides or proteins, Analysis of purified intact proteins (<100 kDa) and peptides, Analysis of protein/peptide mixtures by LC-MS

Protein identification via tandem mass spectrometry (LC-MS/MS) including extensive data base search (Mascot, OMSSA, XTandem, ProteinLynx Global Server, ...) and sample preparation for mass spectrometric analysis (in-solution digestion, desalting)

 

Available support and techniques

Sample preparation

Liquid chromatography-mass spectrometry (LC-MS) method development

Quantitative proteomics (SILAC, Label free, SRM)

Post-translational modification analysis

De novo peptide sequencing (Peptides)

Protein sequencing (Edman N-Terminal protein sequencing)

Applied instrumentation

Reflex IV MALDI TOF (Bruker)

QTOF2 (Micromass, Waters)

LC/MSD Trap XCT Ultra (Agilent)

QTOF Premier (Micromass, Waters) 

Orbitrap Fusion (Thermo Scientific)

Orbitrap QExactive (Thermo Scientific)

 PRICELIST

Research

Mass spectrometric proteomics

The aims of the group are:

Ø  to develop an interactive, multi-disciplinary approach to protein analytical problems, bringing together scientific expertise from diverse areas such as biology, biochemistry bioinformatics and medicine

Ø  to apply advanced proteome analytical methods based on protein and peptide separation methods, liquid chromatography and mass spectrometry

Ø  to further improve proteome analytical methods including sample preparation to meet the needs in biochemistry and medicine,  to teach students, postdoctoral fellows and practicing scientists in proteomics with a focus on the separation of peptides and proteins and their analysis by mass spectrometry.

The term proteome, which was introduced in 1995 by Mark Wilkins [1], is a hybrid from the words protein and genome, thus implicating the description of the entire protein complement encoded by an organisms DNA. There is a huge difference in complexity between the genome and the proteome, because the genome is more or less static in comparison to the proteome, which is continuously changing from the beginning of life of an individual organism until its death, thereby reflecting the different stages of development as well as the interaction of the organism with its environment. The proteome of an organism is many orders of magnitude larger than its genome. It is estimated that the human genome consists of 20,000 to 25,000 genes [2], which code for much more than 500,000 protein species [3].

Proteomics is a term describing the comprehensive analysis of proteomes. Consequently proteomics focuses on the study of proteins: their chemical structures and their functions, concentrations, localisations, interactions with other biomolecules and their lifetimes.

Mass spectrometry is one of the most important tools for the analysis of the chemical composition of proteins. The analysis of proteins by mass spectrometry became possible by the invention of the "soft" ionization and desorption techniques termed matrix-assisted laser desorption/ionization (MALDI), developed by Karas & Hillenkamp [4] and electrospray-ionization (ESI) by developed by Fenn [5]. The inventions of these soft ionization techniques for the analysis of large biomolecules were rewarded with the Nobel Prize in Chemistry in 2002.

Successful mass spectrometric analysis of proteins critically depends on appropriate sample preparation, which has to ensure that the complexity of the composition of the sample is reduced and that all molecules negatively interfering with the mass spectrometric analysis are removed prior to the analysis of the intact protein or its enzymatic cleavage products. For the reduction of the complexity of a biological sample two main strategies have been established in proteome analysis, the bottom up and the top-down approach (Fig. 1). Bottom-up approach: The proteins are either directly  enzymatically digested by an enzyme like trypsin (path 1 in Fig. 1) or separated first by 2-dimensional electrophoresis and then digested gel spot by gel spot (path 2 in Fig. 1). The first approach (path 1 in Fig. 1) requires at least a 2-dimensional separation for reducing the complexity of the peptide mixture. Top-down approach: The proteins of a protein extract are separated firstly and then introduced as intact molecule ions into the mass spectrometer (path 3 in Fig. 1). 

Liquid chromatography is the most important method applied as the final sample preparation step prior to the mass spectrometric analysis.

 


Figure 1: Main strategies for protein identification in proteomics. A deeper discussion of this figure is given under Research Topics.
1: The shot-gun approach; 2: 2-dimensional electrophoresis approach. 3. Mass spectrometric top-down approach. 2-DE: Two-dimensional electrophoresis. LC: Liquid chromatography. MS: Mass spectrometry. md-S: multi-dimensional separation. PMF: Peptide mass fingerprint. PTM: Posttranslational modifications. ID: identity. -: few information; +: identification of a protein species possible. ++: Comprehensive information allows a more or less full description of the exact chemical composition.


 

 

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Letzte Änderung: Anuschka Ollnow, 17.06.2015