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55 Cards in this Set
- Front
- Back
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What does mass spectrometry detect? This allows it to measure what? |
Detects: charged ions in gas phase Measures: mass to charge ration (m/z) |
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The potential mass spectrometry methods can be: EI, CI, FAB, ESMS or MALDI. What does each acronym stand for? |
EI: Electron Impact CI: Chemical Ionisation FAB: Fast Atom Bombardment ESMS: ElectroSpray Mass Spectrometry MALDI: Matrix Assisted Desorption/Ionisation |
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What 2 mass spectronomy methods are most common? |
ESMS and MALDI |
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ESMS and MALDI allow you to detect proteins, nuceli acids and post translational modifications. State 4 other uses of mass spec? (What else can it detect?) |
Answer can include any 4 of the following: Protein sequencing, Modifying nucleic acids, Enzyme mechanics, Protein folding, Proteomics and Non-covalent assemblies |
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There are 4 main steps involved in the process of ionisation for mass spectrometry. Step 1 forms a fine spray of droplets. What property does these droplets have? How is this achieved? |
High surface charge densities, due to high potential on capillary needle |
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Step 2 involves the evaporation of carrier solvent. What does this do to the droplets? |
They shrink, charge density on surface increases |
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Step 3 involves explosive fragmentation. This occurs as the droplets reach what? What change occurs at this point? |
Raleigh limit, droplet surface charge density exceeds surface tension |
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In step 4, desolvation continues. What does this form? |
Gas phase ions, either +ve or -ve. |
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Because ESMS and MALDI can produce multiple charged ions on a single protein, the m/z ratio will be higher. What range is the m/z raio typically in for MALDI and ESMS? |
500-2,000 m/z |
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For ESMS, masses up to what can be observed? |
150,000 Da |
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State 2 advantages of using ESMS? |
Accurate: 1 Da in 10,000 (0.01%) Doesn't require a strong vacuum |
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State one form of ESMS? |
Fourier Transform Ion Cyclotron Resonance (FTICR) |
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What does FTICR produce? |
Deconvolution allows a Guassian distribution (average isotropic mass) |
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State 2 advantages of using FTICR? |
Very sensitive and a very high resolving power |
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For larger proteins, what form of MS is used? |
Matrix-Assisted Laser Desorption Ionisation (MALDI) |
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There are 5 main steps involved in detection using MALDI. State step 1. |
Sample mixed with crystalline matrix and irradiated with a laser |
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State step 2 involved in detection using MALDI. |
Matrix absorbs energy from laser, sample is volatised through pulses |
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State step 3 involved in detection using MALDI. |
Sample molecules pick up protons from the matrix |
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In step 4, an electric field accelerates the ions. For step 5, a high m/z is produced, and so what is measured? Why is this measured? |
Time of flight (TOF), all ions have same KE (=1/2mv^2), heavier ions move more slowly |
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What data would be seen from a MALDI detector? |
1 peak per component |
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State 2 uses of MALDI. |
Rapid ientification of a protein of a known sequence Proteomics |
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For protein sequencing using MS, there are 3 steps involved. State step 1. |
Use precision enzymes or chemicals to produce mixture of peptides broken at known locations. |
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Step 2 is analysis using MS. What 2 forms of analysis can be used? |
ESMS coupled to HPLC Directly via MALDI: provide MW and 15 a.a. in on each fragment |
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State step 3 involved when sequencing a protein using MS. |
Sequencing: Compare massses of fragments with those predicted from genome |
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What enzyme cuts on C terminal of lysine or arginine? |
Trypsin |
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What enzyme cuts on C terminal of Phenylalanine, Tryptophan or Tyrosine? |
Chymotrypsin |
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What enzyme cuts on C terminal of glutamate? |
V8 protease |
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What chemical cuts on C terimal of methionine? |
Cyanogen Bromide |
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What chemical cuts on N terminal of cysteine? |
2-Nitro-5-Thio-Benzoic Acid |
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Of the Post Translational Modifications, what mediates phosphorylation? Where can the OH group come from? |
Kinases, OH group comes from serine, threonine or tyrosine |
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What mass shift is seen with phosphorylation? |
+80 Da |
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What does lysine acetylation control? |
Histone binding to DNA |
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What mass shift is seen with lysine acetylation? |
+42 Da |
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N-linked glycosylation. Oligosaccharides are attached to asparagine through what? |
N-acetylglucosamine |
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What mass shift is seen through N-linked glycosylation? |
+203 Da |
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Cell surface O-linked glycosylation only occurs at 2 amino acids. Name both. |
Serine and Threonine |
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What mass shift is seen with cell surface O-linked glycosylation. |
+203 Da |
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What mass shift is seen with reversable O-GlcNAc-ylation? |
+203 Da |
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On what amino acid can lipidation occur? |
Glycine |
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Lipidation involves the process of what? |
Myristoylation |
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What mass shift is seen from lipidation? |
+210 Da |
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Where can farnestylation occur? What is removed and produced? |
Cysteine, 4 amino acids from C terminal Last 3 amino acids removed, forms a methyl ester |
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What mass shift is seen with farnestylation? |
+235 Da - 3 amino acids |
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If you encounter a brand new protein, there are 3 steps involved. State step 1 and 2. |
Step 1: Trasitional sequencing using fluorophores Step 2: Plan digest enzymes |
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Step 3 involves building a database. What information is included for this database? |
Mass of peptide frangments and the sequence of 15 amino acids in on each fragment |
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State 2 limitations of using MS as a form of protein sequencing? |
Only see 15 amino acids in from terminus Can't distinguish between leucine and isoleucine |
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For protein-ligand binding, what type of binding is needed for it to be sequenced? |
Covalent |
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If a non-covalent bond between protein and ligand, what can be detected in the ligand? |
Bound metals |
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The MS of DNA can be used to observe? |
ssDNA, ds DNA and Quadruplex DNA |
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State 2 Considerations when using MS to observe DNA? |
Answer can include any 2 of the following: Hydrogen bonds don't survive ionisation Every phosphate group on ladder picks up metal in buffer, e.g. NaCl Ion peaks of ssDNA seen instead of dsDNA, carry out at -50 to -15 degrees (cold spray) |
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For ESMS and MALDI, compare the: Ionisation Mass Range |
Ionisation ESMS: continuous MALDI: Pulsed Mass Range ESMS: 1,000,000+Da MALDI: 1,000,000+Da (70,000+ MALDI is better) |
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For ESMS and MALDI, compare the: Detection limit Accuracy Sensitivity to contamination |
Detection limit ESMS: 10^-15 to 10^-21 MALDI: 10^-15 to 10^-18 Accuracy ESMS: High MALDI: Not as good Sensitivity to contamination ESMS: Reduces sensitivity, must be salt free MALDI: Reasonably tolerant (mM of salt tolerated) |
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For ESMS and MALDI, compare the: Spectral simplicity Coupling with online separation Noncovalent interactions |
Spectral simplicity ESMS: Gaussian distribution MALDI: Single peak Coupling with online separation ESMS: HPLC MALDI: Not needed Noncovalent interactions ESMS: Yes MALDI: No |
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Which of the 2, ESMS or MALDI is used for tandem sequencing of proteins? |
ESMS |
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What is tandem MS? |
Involves multiple steps of MS slection, with some form of fragmentation occuring in between |
