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Cancer Research (Subject) / V.8 Proteomics (Lesson)
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- Which kinds of spotting in protein microarrays do exist? •Robotic contact printing or robotic spotting: ‒most common type of protein array ‒robots place large numbers of proteins or their ligands onto a coated solid support in a pre-defined pattern •Ink-jetting: ‒a drop-on-demand, non-contact method of dispersing the protein polymers onto the solid surface in the desired pattern •Piezoelectric spotting: ‒similar method to ink-jet printing, also a non-contact process ‒the printhead moves across the array and uses electric stimulation to deliver the protein molecules onto the surface via tiny jets •Photolithography: ‒a photo-protected quartz substrate is selectively illuminated by light passing through a photolithographic mask; -deprotected areas get activated
- What is a protein microarray and how does it work? A high-throughput method: ‒to track the interactions and activities of proteins ‒to determine their function on a large scale •Consists of a support surface (glass slide, NC membrane, bead, or microtiterplate) to which an array of capture proteins is bound •Addition of probe molecules, typically fluorescently labeled -Any reaction between the probe and the immobilized protein emits a fluorescent signal that is read by a laser scanner -Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents
- How are proteins immobilized on a slide for the protein microarray? The coating of the slide / solid surface enables... -immobilizing the protein and preventing its denaturation ‒orienting it in the appropriate direction, so that its binding sites are accessible ‒providing a hydrophilic environment for the binding reaction (Microwells have the dual advantage of providing an aqueous environment, while preventing cross-contamination between samples) ‒displaying minimal non-specific binding in order to minimize background noise in the detection systems ‒being compatible with different detection systems
- Define proteomics. Why is it so complex? Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics is complex, because...: •the proteome is constantly changing •samples are easier degraded, than DNA samples •Huge range of concentrations (up to 10^6 fold differences) •Level of mRNA not necessarily equal to level of protein product •Some transcripts give rise to multiple products (alternative splicing, etc.) •Many proteins undergo post-translational modifications
- What are the methods used in proteomics? Mass Spectrometry = Main Approach •MUDPIT (Multidimensional Protein Identification Technology) •MS/MS •Protein/Chemical Compound/Antibody Arrays •GFP + FRET, or other fluorescence based approaches
- What are aptamers and how are they created? Aptamers are oligonucleotide or peptide molecules, that bind to a specific target molecule. --> created by selecting them from a large random sequence pool
- What are the three types of protein microarrays? •Analytical microarrays = capture arrays •Functional protein microarrays = target protein arrays ‒constructed by immobilizing large numbers of purified proteins ‒used to identify protein-protein, protein-DNA, protein-RNA, protein-phospholipid, and protein-small molecule interactions, to assay enzymatic activity and to detect antibodies and demonstrate their specificity •Reverse phase protein microarray (RPPA) ‒involve complex samples, such as tissue lysates
- Which are the five major areas of application of protein microarrays? -Diagnostics -protein expression profiling -Protein functional analysis -Antibody characterization -Treatment development
- What are the challanges of protein microarrays? 1) finding a surface and a method of attachment that to maintain their secondary or tertiary structure 2) producing long shelf life 3) identifying and isolating antibodies against every protein in the human genome 4) quantifying bound protein while assuring sensitivity and avoiding background noise 5) extracting the detected protein from the chip in order to further analyze it 6) reducing non-specific binding by the capture agents, 7) sufficient capacity of the chip to allow complete representation of the proteome
- What does Mass Spectrometry measure? Mass Spectrometry measures molecular or atomic weight.
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- Beschreiben Sie die einer Massenspektrometrie vorangehenden Schritte. Step 1: Isolate cells or other protein source Step 2: Lyse cells and isolate proteins Step 3: Break up proteins into smaller (but still relatively large) amino acid chains Step 4: Separate chains (2D gel, gas or liquid chromatography) --> Step 5: Analyze separated protein parts by mass spectrometry
- Nennen Sie zwei Ionisierungsquellen bei Massen Spektrometrie. (Abkürzung und ausgeschrieben) -ESI = Electrospray Ionization -MALDI = Matrix-assisted Laser Desorption / Ionization
- Which types of mass analyzers do exist? 1) Ion Trap •Sensitive, extremely fast •Low resolution and mass accuracy 2) Orbitrap: high resolution and mass accuracy (incl. first stage linear ion trap) 3) Time of Flight (TOF) •Sensitive, high mass accuracy, high resolution •Limited to small m/z ratios •Not easily coupled to chromatography 4) Quadrupole •Sensitive, acceptable mass accuracy and resolution •Easily coupled to chromatography 5) Fourier Transform ion cyclotron •High sensitivity, mass accuracy, resolution, dynamic range •Expensive, difficult to operate, low fragmentation efficiency
- What is the difference between label and label-free quantification? SILAC (stable isotope labeling by/with amino acids in cellculture) is a technique based on mass spectrometry that detects differences in protein abundance among samples using non-radioactive isotopic labeling. Label-free quantification may be based on precursor signal intensity (for high precision mass spectra, such ToF, fourier transform ion cyclotron resonance (FTICR), or Orbitrap) or on spectral counting.
- What does a mass spectrometer consists off? A mass spectrometer consists of an ion source, in which the sample molecules are ionized, a mass analyzer, which separates the ions according to their mass-to charge ratio (m/z), and a detector that records the ions. A sophisticated computer system controls the mass spectrometer and the data-dependent acquisition functions.
- What happens in a MS/MS PTM´s detection? 1) In the mass spectrometer an ion source ionizes the sample molecules. 2) A mass analyzer separates the ions according to their mass-to charge ratio (m/z) 3) The detector records the ions. 4) A computer system controls the mass spectrometer and the data-dependent acquisition functions. --> MS spectrum acquired 5) Peptides are in turn selected for MS/MS. [Fragmentation of the peptide amide bond produces a set of fragment ions that generate a ladder-like readout of the sequence in the tandem mass spectrum. The presence of a PTM will change the mass of the modified amino acid residue and of the peptide.] --> mass of the PTM and the identity and position of the modified amino acid residue are revealed
- Which methods of PTM enrichment exist and how do they work? -Affinity enrichment --> tissue fractionation -> digestion -> using an antigen against the PTM, that is conjugated to beads / antibody-> elution -> analyses [Nano LC/LC or MS/MS] Covalent capture --> solid support like beads/matrix -> chemicals covalently link the PTM to the solid support -> enrichment of the modified proteins on solid support
- Name known methods for protein separation. -Protein Gel electrophoresis -SDS-Page (SDS -POLYACRYLAMIDE GEL ELECTROPHORESIS) -2D Gels (TWO-DIMENSIONAL GEL ELECTROPHORESIS) -ISOELECTRIC FOCUSSING -LIQUID CHROMATOGRAPHY -> Gel filtration chromatography -> Ion exchange chromatography -> antibody-affinity chromatography
- What does HPLC-MS stand for? High pressure liquid chromatography coupled with Mass spectrometry -> combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS). High sensitivity and selectivity.
- Which are the 4 scan modes of MS/MS? 1) precursor (parent) ion scan [precursor ion in the 1. mass analyzer, product ion in 2.m.a.] 2) product (daugther) ion scan [precursor ion in the 1. mass analyzer, all others after 2.m.a.] 3) neutral loss scan [all masses in the 1. mass analyzer, 2.m.a. scans offset masses] 4) selected reaction monitoring [1.+2.m.a. scan selected masses]