MS Flashcards
Why is sequencing proteins relevant? (5)
Knowledge of the sequence of a protein is usually essential to elucidating its mechanism of action (e.g., the catalytic mechanism of an enzyme).
Proteins with novel properties can be generated by varying the sequence of known proteins.
Amino acid sequences determine the three-dimensional structures of proteins.
Sequence determination is a component of molecular pathology, a rapidly growing area of medicine.
The sequence of a protein reveals much about its evolutionary history.
What is the first step in protein sequencing?
Proteins need to be digested into peptides (linear): break intermolecular interactions
How are proteins digested into peptides?
- Protein reduction
- Protein alkylation
- Protein digestion
Why are proteins reduced?
To break disulphide bonds, which stabilise tertiary structure of protein (only strong, covalent interaction involved in this)
What chemical is used to reduced disulphide bonds?
Dithiothreitol, used in excess
Why is protein alkylation importants in order to digest them into peptides?
Once chains have been reduced, one more step is needed to ensure that the disulphide bonds do not reform
What chemical is used for protein alkylation?
Iodoacetate, gives separated carboxymethylated chains on Cys
What is the most commonly used enzyme used in protein digestion?
Trypsin, a serine protease
What is the function of trypsin and its specificity?
Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins.
It is produced in the pancreas as the inactive proenzyme trypsinogen.
Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline
Why is trypsin so widely used in protein digestion?
highly specific
efficient
generates peptides having an arginine or lysine at the C-terminus (which simplifies sequencing a whole lot)
What is the specificity of chymotrypsin?
It cleaves the carboxyl side of the amide bond (the P1 position) is a large hydrophobic amino acid (tyrosine, tryptophan, and phenylalanine)
What are the essentials of a MS?
What is the best way of ionising a sample for MS or Tandem MS?
ESI
How does ESI work?
Protein solutions are passed through a metal needle held at high potential (typically 3-5 kV)
There is a build-up of charge at the needle tip which overcomes the surface tension producing a fine mist of droplets that emerge from the needle tip with a net charge.
A counter current drying gas aids desolvation and droplet evaporation to produce smaller daughter droplets.
This eventually leads to the production of solvent free molecular ions
Why are charged particles, with both m and z, needed for MS?
Because they can be handled and their paths controlled thru the use of electric and magnetic fields.
Are all ions produced +vely charged?
Generally, yes, but there are important exceptions (NA)
Describe the mass spectrum
Ions appear in a spectrum as a result of an electrical current that is generated and amplified when the ion strikes the detector
X-axis is the m/z ratio of the ion
Y- axis is the ion current or more commonly the relative ion abundance
Sometimes base peak is normalised to 100% and all other ions have abundances relative to this peak
What is MS/MS?
Means by which to obtain the MS of an MS, by causing ions in the first MS to fragment so that the secondary MS can reveal new information
What are the main components of a tandem mass spectrometer and their functions?
Ionisation source: ionises sample so that it can be handled
MS1: separates ions based on m/z value, isolates ion of interest
Collision cell: activates and dissociates ion of interest
MS2: separates and detects fragment ions
Detector: detection of ions, production of spectrum
What is CID and what can it be used for?
If an ion collides with a neutral atom (usually a noble gas) or molecule, some of its translational energy is converted into internal energy (bond vibrations) causing the ion to fragment
CID can be used to fragment peptides and therefore obtain their sequence
What are the components of the Q-TOF analyser?
What is the function of the quadrupole in Q-TOF?
MS mode: guide for ions
MS/MS: can be set to allow only the ion of interest to pass into the collision cell where fragmentation is induced. Fragments are then analysed in detector
What does the detector do in Q-TOF?
Converts ion signal from e- to photons and back to e-, it is a photomultiplier tube
What are the three different types of bonds that can fragment along the amino acid backbone?
NH-CH , CO-NH, CH-CO
Each bond cleavage gives rise to two species, one neutral and the other one charged. The charged species is the one detected by the mass spectrometer
How many possible fragment ions are there for each amino acid residue?
6 (a,b,c,x,y,z)
a,b,c ions retain charge on which terminus?
the a, b, and c ions have the charge retained on the N-terminal fragment
x,y,z ions retain charge on which terminus?
x, y, and z ions have the charge retained on the C-terminal fragment
Which is the most common cleavage site and what ions arise from this?
CO-NH (amide linkage), gives rise to b and y ions
What are a ions?
b ions - C=O group
What are c ions?
b ions + NH group
What does the intensity of the signal in an mass spectrum reflect?
how often that bond breaks
Which peak gives the Mr of the ion?
The one with the biggest m/z value, gives [M-H]+
What is the residue mass for an amino acid?
Residue mass = Amino acid mass – H2O
What is the structure of an immonium ion and why are these useful?
Immonium ions appear in the very low m/z range of the MS/MS spectrum but some are of low abundance.
Most amino acid residues yield a diagnostic immonium ion, but. The immonium ions are useful for detecting and confirming many of the amino acid residues in a peptide, although no information regarding the position of the residues in the peptide sequence can be derived from immonium ions.
What is the difference in mass between an a and a b ion?
a ions are b ions that have lost a CO group
An a ion has a mass of 28 Da less than the corresponding b ion
What are the masses of a,b and y ions?
[N] = mass of the N-terminating group (H = 1)
- [C] = mass of the C-terminating group (OH = 17)
- [M]n = sum of the masses of n amino acid residues (+ any PTMs)
How is the probable number of resiudes found?
Divide peak with highest m/z value by 110, which is the average residue mass
Identify a,b,c,y,x,z ions in a sequence
