Mass Spectrometry Flashcards
why sequence proteins?
knowledge of the sequence is essential to elucidating its mechanism of action
amino acid sequences determine the three-dimensional structure of proteins
the sequence of a protein reveals much about its evolutionary history
what is trypsin?
serine protease
found in the digestive systems of many vertebrates
produced in the pancreas as the inactive proenzyme trypsinogen
cleaves pepetide chains mainly at the carboxyl side of lysine and arginine, except when these are followed by proline
highly specific and efficient
what is mass spectrometry used for?
biotechnology - analysis of proteins, peptides, oligonucleotides
pharmaceutical - drug discovery, combinatorial chemistry,pharmacokinetics, drug metabolism
environmental - water quality, food contamination
geological - oil composition
materials - polymer microcomposition
clinical - neonatal screening, drug testing
what is the sequence of events that occurs in a mass spectrometer?
sample introduction –> ionisation source –> mass analyser (separates ions based on m/z ratio) –> collision cell (dissociates ions) –> mass analyser –> ion detector –> computer system
describe the method of electrospray ionisation mass spectrometry
- a solution of analyte passes through a charged needle, kept at a high electric potential
- solution is dispersed as a fine mist of charged microdroplets
- the solvent surrounding the macromolecules rapidly evaporates, leaving multiply charged macromolecule ions in the gas phase; this is aided by a countercurrent drying gas
- protons addedduring passage through the needle give additional charge to the macromolecule
why can’t we use mass spectrometry to analyse macromolecules? what method do we use instead?
mass spectrometry analyses molecules in the gas phase; macromolecules are easily decomposed by heat
instead, we use electrospray ionisation mass spectrometry (ESI MS), in which macromolecules are forced from the liquid to the gas phase
what is tandem mass spectrometry used for?
Tandem mass spectrometry is used to obtain the mass spectrum of a mass spectrum by causing ions in the first mass spectrum to fragment so that the second mass spectrum can reveal new information.
what is collision-induced dissociation?
When an ion collides with a neutral atom or molecule, some of its translational energy is converted into internal energy (bond vibrations), causing the ion to fragment. In the collision cell, argon or helium is often used.
what are the roles of the quadrupole and TOF in mass spectrometry and tandem mass spectrometry?
MS - qudrupole operated in RF-only mode and so acts as a guide and does not separate ions; ions separated in TOF analyser
MS/MS - quadrupole can be set to allow only ion of interest to pass into the collision cell; fragments are then analysed in the TOF
what is the quadrupole?
a mass analyser consisting of four cylindrical electrodes set parallel to one other that applies DC and alternating fields that act together as a double filter to determine the specific m/z ratio of ions allowed to pass through into the collision cell
what is TOF MS?
TOF MS stands for Time-of-Flight Mass Spectrometry. An extraction field is applied to accelerate the ions. Since the ions are of different masses, they require the same energy to accelerate but will travel at different velocities. The masses of the ions can be analysed by recording flight time.
which ions retain charge on the N-terminal fragment?
a/b/c ions
which ions retain charge on the C-terminal fragment?
x/y/z ions
how is the residue mass calculated?
residue mass = amino acid mass - H2O
since amino acids lose a water molecule when a peptide bond is formed
what is an immonium ion?
H2N+=CHR
note that no information can be obtained from the immonium ion regarding the position of the residue
