L2: Mass Spec Flashcards
Human genome size
20,000-25,000 genes
3.15x10^9 bases
True or false:
Post-translational modifications are directly coded by genes
False.
If a protein has 50 known modifications, how many possible proteoforms exist?
50! or 3.04e64
Y axis of mass spec measures what?
Intensity
X axis of mass spec measures what?
mass/charge (m/z)
Mass spectrometers can routinely detect ____ moles of sample
10^-15
MS is used to detect what?
mass, identity, sequence or composition (Tandem MS), and PTM of a peptide
Mass spectrometers work by pushing a) _____ using b) _____ fields.
a) gas phase ions
b) electric or magnetic
When working with proteins and peptides, which two techniques are by far the most common methods for generating gas phase peptide/protein ions?
Electrospray and MALDI
Basic components of a mass spectrometer
- Source (e.g. MALDI): ionises sample into ‘flight’
- Analyser (e.g. quadrupoles or time-of-flight): separates sample based on m/z
- Detector (e.g. electron multiplier): detects ions
- Computer: deconvolutes data and plots as abundance vs. m/z
Time of flight analyser
- Ions accelerate to a detector with the same energy (E)
- Smaller ions reach the detector 1st
- Require a good vacuum (<10-6 torr)
- Simple but expensive
- Mass range up to 500 kDa
- Mass determined according to equation: m = 2E/v^2
Quadrupole analyser
- Four parallel rods with a DC voltage and a superimposed AC (RF) potential
- Scan DC and RF to allow ions of increasing m/z to successfully pass to a detector
- Can capture specific ions and eject specific ions
Advantages and Disadvantages of Quadrupole vs. ToF
Quadrupole advantages:
- Tolerant of poor vacuums
- Low cost
Disadvantages:
- Low resolution
- Analyse ions m/z <2000
Orbitrap Mass Analyser
- DC electric field causes trapping of ions
- Ions oscillate radially around centre electrode and axially along z axis
- Ions are non-destructively detected via their z oscillation frequencies
- High mass resolving power (450K) and mass accuracy (1ppm)
Tandem MS (MS/MS)
- Tandem MS analysers have 2 or more analyser chambers
- Some analysers can perform MS^n by re-trapping ions
- Peptide bonds are broken usually by Collision Induced Dissociation (CID) in the collision cell (argon or helium)
MALDI-TOF Mechanism
- Sample to be analysed is mixed with a matrix compound
- Solvents vaporise, leaving only the recrystallised matrix, but now with analyte molecules spread throughout the crystals. Matrix and analyte are said to be crystallised in a MALDI spot
- Matrix has a strong optical absorption in either UV or IR range so that they rapidly and efficiently absorb the laser irradiation.
- The Matrix is often acidic, acting as a proton source to encourage ionisation of the analyte.
MALDI-ToF effective mass range
500Da to 1,000,000+ Da
Electrospray ionisation (ESI)
- Most common source in HPLC, known as a soft ionisation technique (intact molecules are detected).
- Solvent must be removed to produce gas phase ions
- Must only use mobile phase additive which are volatile
- Protein in droplets is stranded in gas phase and sucked into the spectrometer
Biologists often submit samples in buffer and other
salts for analysis. What happens if you spray salt
solutions?
Electrospray requires your sample to be in a volatile solvent and not contain salts and other non volatile components as salt will crash out inside the source of the MS, corroding the MS source
Why is HPLC used in MS?
To separate out the salt, and separate the protein.
Sample goes through HPLC and is injected onto an rpHPLC column. The protein sticks to beads inside the column and is eluted with acetonitrile gradient
The same molecule with 2 charges will reach the MS detector in a) HALF / DOUBLE the time as it experienced b) HALF / DOUBLE the force
a) Half
b) Double
Difference between monoisotopic and average mass
Monoisotopic mass is used for small molecules
Average mass is used for big proteins
Trypsin cuts at which amino acids?
Lysine (K) and arginine (R)
Basis for peptide mass fingerprinting
- List of peptide masses is generated
- Values of peptide masses are inserted into an algorithm e.g. MS-Fit
- A protein is matched from a genome database to correspond to the masses based on known protein masses.
Advantages and disadvantages of peptide mass fingerprinting
Advantages:
• Fast and sensitive
Disadvantages:
• Must have sequence in database
• Need to know which enzyme was used to digest fragment
• Protein can’t be heavily modified
In an MS/MS ions search, fragmentation of the backbone occurs at which bond?
Peptide bond
Most common ions produced by CID are what?
b and y ions
How to find first y ion in a tryptic digest?
Look for
lysine = 128 +19=147
arginine = 156+19=175
Largest protein database
uniprot but lots of redundancies
Database with no redundancies
MSDB
