Proteomik
Commassie Brilliant Blue Advantages/Drawbacks?
Advantages:
* Cheap
* Low background staining
* Easy to use
Drawbacks:
* Not very sensitive
* Limited dynamic range
* Glu sides may be modified
Silver staining Advantages/Drawbacks?
Advantages:
* Good detection limit
* High sensitivity
Drawbacks:
* Sometimes high background staining
* Some proteins may not be stained
* Labour intensive
Non-covalent fluorescent labeling Advantages/Drawbacks?
Advantages:
* Sensitive
* Low background staining
* High dynamic range
* All proteins stain well
Drawbacks:
* Expensive
Difference between proteomics and classical protein chemistry?
Proteomics:
* Holistic approach - "top down". Understanding system.
* Studies of "all proteins at the same time" and their role in the biological network. Patterns
* Emphasis on identification and quantification.
* Discovery driven research.
* Enormous amount of data
Classical protein chemistry:
* Reductionist approach - "bottom up". Understanding components.
* Detailed studies of individual proteins.
* Emphasis on structure and function and the relation between them.
* Hypothesis driven research.
* Small amount of data
Next generation Sequencing
* Is used for mRNA quantification
The principle:
* DNA polymerase adds fluorescent nucleotides one by one onto a growing DNA template strand, and each incorporated nucleotide is identified by the fluorescent tag.
Example of a popular NGS - Illumina Solexa
1. Sample preparation
2. Cluster generation
3. sequencing
4. Data analysis=> quantification
RNA-Seq by illumina solid-phase sequencing
* Adaptor sequence are ligated to the ends of each cDNA fragment.
* The sample is introduced via a flow cell to a glass slide, where oligonucleotides complementary to the adaptor sequence are attached =>immobilization of the cDNA fragments.
* Clusters of cDNA clones are formed on the surface by bridge amplification.
* 50 millions cluster in each flow cell => parallel analysis!
* Synthesis is perfomred using uniquely, fluorescent labeled dNTPs.
* Each clusters is monitored after the incorporation of each dNTPs by laser illumination to determine of A, C, T or G had been incorporated. This is done for all clusters simultanously.
* In this way, the sequence of the cDNA in each cluster is revealed.
* If an mRNA sequence was present in many copies in the original sample, many clusters with that sequence will be present on the slide => allows for quantification of that mRNA, relative to all other mRNAS represented on that slide.
DNA Arrays
* Gene-specific DNA strands (probes) are distributed in individual spots on a chip
* Labelled cDNA, prepared from mRNA from the sample under study, is added.
* Hybridization is detected by fluorescence.
* Comparison of sample (healthy/diseased)
DNA arrays - Spotted arrays
* ds cDNA strands that have been amplified by PCR are distributed on glass chip or nylon membrane via spotting
* Denaturation (dehybridization) is required before analysis
Multiplex assay:
* Comparing expression levels in different samples (e.g healthy/disease)
* Samples are covalently labeled with different fluorophores , for excitation and emission at different wavelength.
* Labeling: cDNA is made from the mRNA in the sample, using fluorescently labeled nucleotides.
* Samples are mixed and added to an array for hybridization.
* The array is scanned twice, exciting the two different fluorophores.
* The combined image reveals differenlty expressed genes.
DNA-arrays - Oligonucleotides arrays
* about 20-25 nt
* ss oligodeoxynucleotides are synthezides directly onto the chip
* Each selected probe sequence should be checked against the entire genome in order to avoid cross hybridization
* Many different probes/gene are used in order to obtain more reliable results
* Silicon chip funtionalized with photosensitive groups. Apply a mask with holes where you want to attach the first nucleotide, illuminate to deprotects, and allow reaction with the first nucleotide, which is also derivatized with a photosensitive group. This is done for "first positions" of all probes on the chip. Then repeat for position 2,3,4 etc
Interaction between Biotin and streptavidin
* The interaction between biotin and streptavidin/avidin is very strong and can be considered irreversible.
* The biotin/streptavidin/avidin system is excessively used for high affinity immobilization, affinity chromatography, detection in immunoassays and protein chips, and many other application in proteomics and other fields within the broad area of chemical biology.
Comparison between DNA Array and RNA-seq for mRNA quantification
Novel transcripts:
* RNA-seq methods do not require species- or transcript specific probes. It can therefore detect novel transcripts not known to be present on the sample beforehand. (RNA-seq methods are open while oligonucleotides arrays are closed)
Dynamic range:
* RNA-seq technology can quantify expression across a larger dynamic range than DNA arrays (>10^5 for RNA-seq compared to 10^3 for arrays)
Sensitivity:
* RNA-seq technology can detect genes with even lower expression than DNA-arrays.
Northen blot
mRNA from the sample under study is separated electrophoretically and blotted onto a membrane, followed by addition of different, radiolabeled gene-specific cDNA probes
This technology is used when looking for a few, identified mRNAs but not suitable for large-scale gene expression analysis.
MS: Why are peptides used instead of whole proteins?
* Most instruments have an upper limit of reliable mass detection due to stability and insufficient ionization of larger proteins.
* The intact protein would only give one peak, which is not enough for a reliable identification
Phosphorylation: Give example on what makes experimental identification complicated.
* The modified and the unmodified form of the protein often exist at the same time but the modified is usually at a low concentration.
* Modification can prevent cleavage and modified peptides are often more difficult to ionize.
* To assess all possible phosphorylation sites in a protein full sequence coverage is needed.
Glycans: Why is it more difficult to use ESI-MS/MS than phosphorylation?
Glycans can be very different in size and composition, while a phosphorylation event is just the addition of a small phosphate group which always has the same chemical structure. In an MS/MS spectrum, fragment ions from both the peptide backbone and the glycans will be found, making the spectrum very complex.
Shotgun proteomic approach
* A human cell expresses around 10 000 proteins
* The protein copy numbers span at least seven orders of magnitude
* Cellular core functions are carried out by relatively few proteins, which are present at high abundance, housekeeping proteins - needed in every cell type.
* Regukatory functions are ofter orchestrated by large protein families existing in variable but predominantly low abundance.
* The fraction of total protein mass devited to regulatory functions is higher in human and mouse cells, compared to bacteria and yeast.
* When comparing 11 human cell lines, 73 % of the proteins were expressed in all cell types.
* However, there eas a big difference in expression levels in most of the proteins
* Even many of the most highly expressed housekeeping proteins showed significant differences between cells
What happens in the CID process?
CID induces fragmentation of selected peptide ions. The selected peptides are allowed ti collide with neutral molecules (often helium, nitrogen or argon). In the collision some of the kinetic energy is converted into internal energy ehich results in bond breakage and the fragmentation of the ion into smaller fragments. These fragment ios can then be analyzed by MS/MS.
Give a thorough explanation about y ions.
Y ions (and b ions) are formed in CID. They are defined from the C-terminus (the c-terminal fragment will carry the charge formed upon breakage of the backbone) and the number indicates how many amino acids that form the y ion (y5= 5 amino acids). The purpose of finding the y-ions is to sequence the peptide in order to make a databased-based identification.
Two-hybrid assay vs TAP
* TAP uses endogenous promoters, which means that protein concentration are not artifically high as in two-hybrid assays, where expression in from plasmid. As interactions are concentration-dependent, this can be a source od discrepancy between the studies.
* If the reporter gene is not properly acctivared because of steric hindrance (the proteinsmay interact but the ativation domain may not be in the correct position to stimulate transcription) there will be a false negative result in the two-hybrid assay. With TAP, no reconstitution of a transcription factor is necessary so here the interaction may register.
* Weak interaction may be invisibe in the TAP experiment because weakly interacting proteins may be washed away in the affinity chromatography step. In the two-hybrid assay, these interactions can have a better chance of being observed.
* In both experiments, fusion proteins are used, bur in a different way. The TAP-tagged protein is indeed a fusion protein, but it will not be the same fusion as when this protein is expressed with the activation domain or binding domain of transcription factor. Moreover, the interaction partners of the TAP-tagged protein will not be fusion proteins at all. These differencex can be a source of different results with the two methods, as the type of fusion can affect folding and accessibility to binding sites of the proteins.
Surface preparation: protein chip - strategies to overcome challenges
* Strategies to preserve the native protein structure:
Different types of surface modification:
- hydrogels, such as dextran or polyethylene glycol
- organic monolayers (e.g alkane thiolate coating on noble metal surface
* Strategies to increase protein accessibility:
- Direct immobilization via His-tag, GST-tag, protein A etc
- Immobilization in a hydrogel providing a native-like "3D environment"
* Strategies to reduce non-specific analyte-surface interaction:
- Surface modification, e.g hydrogel or monolayers
- "blocking" of the surface by addition of e g milk och bovine serum albumin (BSA) before analyte application.
- Additives in the analyte solution