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Workflows
Here we outlined some of the workflows for protein/proteome analysis available at the Centre for Protein Research.
- Protein identification from gel bands/spots
- High throughput (shotgun) proteomics
- Quantitative proteomics
- De novo sequencing
- Analysis of PTMs
- Intact mass measurement and sequence tag analysis
Protein identification from gel bands/spots
Protein bands/spots of interest are excised from the gel and digested in-gel with a site specific protease such as trypsin. The tryptic peptides are eluted from the gel matrix and subjected to mass spectrometric analysis. Single protein bands/spots are usually analysed by MALDI tandem Time-of-Flight mass spectrometry. Alternatively LC-coupled tandem mass spectrometry can be performed to gain good sequence coverage and identify other co-migrating proteins or post translational protein modifications. For low complexity samples we usually perform nano-flow RP-LC coupled LTQ-Orbitrap mass spectrometry with a very short LC-gradient.
Proteins are then identified by matching the measured collision induced dissociation (CID) pattern of tryptic peptides with the calculated fragmentation pattern of tryptic peptides predicted by an in silico digestion of the interrogated sequence database. We usually use Mascot and SEQUEST search engines to interrogate various sequence databases. Either amino acid or nucleotide sequences can be used.
High throughput protein identification/proteomics
The goal of high throughput proteomics is basically the identification of all proteins in a certain protein complement including their post-translational protein modifications and quantitative information (see below). It can be performed either as a gel-based or a gel-free approach.
The gel-based approach includes the pre-fractionation and purification of proteins by 1-dimensional protein gel electrophoresis. The gel is then fractionated into several molecular weight fractions to reduce sample complexity and proteins are in-gel digested with trypsin. The tryptic peptides are extracted from the gel matrix and further fractionated by 1- or 2-D liquid chromatography. The liquid chromatography is coupled either off-line to MALDI-based or in-line to ESI-based mass spectrometry.
In a gel-free approach the sample complexity should be reduced on the protein level by various fractionation strategies such as a serial protein extraction followed by liquid chromatography. The proteins are then digested in-solution and the proteolytic fragments further fractionated by 1-D RP-LC or 2-D chromatography using SCX (strong cation exchange chromatography) in combination with RP (reversed phase)-LC. The liquid chromatography is coupled either off-line to MALDI-based or in-line to ESI-based mass spectrometry.
Quantitative proteomics
2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE)
In 2-D PAGE protein complements of two or more different samples are compared by their relative staining intensities after in-gel detection of proteins e.g. by fluorescent dyes or colloidal coomassie. Differences between two samples are excised from the gel and in-gel digested with a site-specific protease such as trypsin. The tryptic peptides are then extracted from the gel and subjected to tandem mass spectrometry (usually MALDI TOF/TOF MS) for protein identification.
Alternatively a 2-D PAGE/MALDI TOF/TOF MS experiment can be extended to a large scale approach to create a 2-D protein reference map. Therefore all visualise protein spots are excised and subjected to mass spectrometry-based protein identification. The reference map is then used to compare protein abundances in various gels of different related samples by matching the positions of protein spots to the identified proteins of the reference map without any further need for MS-based protein identification. This approach gives a more comprehensive picture of the relative protein abundances e.g. of whole pathways or protein complexes.
Isotop-coded affinity tagging (ICAT)
ICAT is a duplex protein labelling technique to differentially compare relative protein abundances of two samples directly with the mass spectrometer. The ICAT chemistry labels the cysteine residues of proteins. Each sample is labelled either with a light or a heavy label that differ in mass by 9 Da. The mass difference of light and heavy label is due to a different carbon isotope composition of both tags and therefore does not alter the chemical properties of the labelled proteins. After labelling both protein samples are pooled, digested with trypsin and further processed for mass spectrometric analysis.
The relative abundances of differentially labelled proteins can be calculated by the relative peak intensities of tryptic peptides labelled with light versus heavy tag that differentially appear in the mass spectrum with an increment of 9 Da.
The ICAT tag also contains a biotin tag for reducing sample complexity by affinity purification of labelled peptides. ICAT is therefore particularly suitable for the quantification of proteins in very complex protein mixtures.
iTRAQ (isobaric tag for relative and absolute quantitation)
iTRAQ is a multiplex protein labelling for mass spectrometry-based protein quantification. It is available as 4-plex and 8-plex labels (we only use 4plex labelling at present). iTRAQ reagents label primary amines (N-terminal amino group and epsilon amino group of lysines) either of intact proteins or proteolytically digested proteins. After labelling samples are pooled and further processed for mass spectrometric analysis.
The iTRAQ labels are isobaric and can not be distinguished by MS only. All differentially labelled peptides of the same species are merged in one peak. Upon collision induced dissociation the tags release low mass reporter fragment ions with a tag-specific mass of either m/z 114, 115, 116 and 117 for the 4-plex label. The relative peak intensities of the different reporter ions in the CID (collision induced dissociation) MS/MS spectrum are used for relative protein quantification.
De novo sequencing
Not all organisms that are of significance for research are completely sequenced yet i.e. conventional mass spectrometry-based approaches for protein identification may fail. These approaches are based on the correlation of mass spectrometric data with predicted data from sequence databases.
Unknown proteins without sequence information in databases can also be sequenced with the mass spectrometer. In general this requires a manual interpretation of MS/MS-data. However the manual interpretation is error prone and many CID (collision induced dissociation)-spectra do not provide unambiguous sequence information. The available software tools for de novo interpretation of CID MS/MS-spectra are as error prone as manual interpretation.
Edman degradation and amino acid detection is still one of the most reliable methods for de novo sequencing of peptides/proteins. However, it is less sensitive than mass spectrometry, labour-intensive and expensive.
Chemically Assisted Fragmentation is a technique that generates high quality sequence data by MALDI-based tandem mass spectrometry. It is based on a chemical modification of the N-terminal amino group. The modification is negatively charged and suppresses b-ions upon fragmentation by PSD (Post Source Decay) or CID in positive ion mode. The fragment spectra contain only y-ions which simplifies the manual interpretation. We are using a different two step chemistry to firstly block the epsilon amino group of lysines by a guanidination (conversion of lysine to homoarginine) and secondly modify the N-terminus of tryptic peptides by a sulfonation with 4-sulfophenyl-isothiocyanate (SPITC). The method is described in Chen et al. (2004) Rapid Commun. Mass Spectrom, 18:191-198. High quality CID-spectra of SPITC-modified peptides reveal unambiguous sequence information by a clear y-ions series. This approach is faster and more sensitive than Edman sequencing.
Analysis of PTMs
Many post-translational protein modifications (PTMs) can be identified by mass spectrometry. Modern high accuracy mass measurements give more confidence in the identification of these modifications. The resolution and mass accuracy of an Orbitrap instrument enables us to distinguish between phosphorylation vs sufation (delta mass of 0.0095), tri-methylation vs acetylation (delta mass of 0.036) or deamidation vs the 13C peak of the unmodified form (delta mass of 0.019) etc.
In general a single protein species is differentially modified i.e. each modification may occur in different ratios and at different sites which complicates the analysis. The abundance of tryptic peptides containing PTMs is therefore lower than the abundance of tryptic peptides that never gets modified. Finding a particular PTM is sometimes as difficult as fining a needle in a haystack and often involves time-consuming manual interpretation of LC-MS/MS data.
Intact mass measurement and sequence tag analysis
Electrospray ionisation generates multiple charge states of large molecules that are detectable in the lower m/z region of the MS-spectrum. A single charge state (up to ca. 20+) can be trapped in the linear ion trap (using an isolation width of 5-10) and transferred to the orbitrap for an accurate mass measurement. At a resolution of 100,000 the orbitrap can resolve the isotope envelope for the determination of the charge state. If the sequence of the protein is known the isotope composition of the most intense peak of the isotope cluster can be calculated and used for accurate mass determination. The error is significantly less than 1 Da for proteins up to 50 kDa. However, only purified proteins or low complexity samples are suitable for this approach.
Top-down approach: In a top-down approach proteins are identification by the mass determination of intact proteins and sequence tag analysis. Therefore a charge state of an intact protein is trapped in the linear trap and fragmented by CID (collision induced dissociation). A high resolution measurement of the fragment ions in the orbitrap allows the identification of doubly or triply charged daughter ions that can be used as precursors for an additional CID fragmentation in the linear trap. This MS/MS/MS analysis of intact proteins requires a reasonable amount of material but allows the identification of peaks of interest in more complex samples.
Electron transfer dissociation (ETD) is a more efficient fragmentation for larger molecules and higher charge states and is probably the best method for top-down protein analysis. Unfortunately ETD is yet not available at the Centre for Protein Research.
