Mass Spectrometry Flashcards
Mass Spec
Involves the detection of ions in the gasphase
The mass is not measured directly, the mass-to-charge ratio(m/z) is measured
Mass spec methods
Electron Impact (EI)
Chemical Ionisation (CI)
Fast Atom Bombardment (FAB)
All unsuitable for biopolymer analysis as they use too much energy to induce ionisation
Electrospray
MALDI
Mass spec uses
accurate mass determination of proteins/ nucleic acids and post-translational protein modifications
protein/nucleic acid sequencing
modified nucleic or amino acids
enzyme mechanisms
non-covalent assemblies
protein folding
proteomics
Electrospray Mass Spectrometry Instrumentation
Electrospray Ionisation Mechanism
The formation of gas phase molecular or pseudomolecular(multiply charged ions takes place in four steps:
- The formation of a fine spray of dropletswith relatively high surface charge densitiesdue to the high potential on the capillary needle.
2 .Evaporation of the carrier solvent molecules from the droplets which causes the droplets to shrink and the charge density on the surface to increase
- Explosive fragmentationof the droplets as the charge density on the droplet reaches a critical limit (Raleigh limit– point at which the droplet surface charge density exceeds the liquid’s surface tension)
- The desolvationprocess continues until the eventual formation of gas phase ionsof individual molecules
Electrospray Ion Detection
Electrospray sources can generate and analyse either positive or negative ions.
• permits the analysis of biomolecules with a wide range of pKa values.
m/z ratio typically 500-2000 Da
Electrospray Ion Detection- Quadrupole Analysers
- Reasonable mass to charge range (up to 4000) - proteins with masses up to 150,000 Da can be observed
- Robust, easy to tune, do not require a very high vacuum (ideal for general lab)
- Resolving power – 1 Da in 10,000
- ions striking the detector transfer charge that registers as a current that is proportional to ion abundance (larger the detector current, the greater the ion abundance)
Electrospray Ion Detection - FTICR (Fourier Transform Ion Cyclotron Resonance)
Extremely sensitive and extreme resolving power
Eg. A crude preparation of red blood cells was used to determine the molecular weight of carbonic anhydrase.
ca. 9 X 10 –18 moles of the enzyme were present MW determined to be 28780 ±1 Da
Resolving power 60,000
information from sequence specific fragment ions confirmed identity of protein from the database
Electrospray Data Interpretation
The conversion of a series of multiply charged peaks into a real mass spectrum is called a deconvolution
- Guassian Distribution
- Tops of the peaks
represent the average isotopic mass
Matrix-Assisted Laser Desorption Ionisation
- Sample mixed with a crystalline matrix and irradiated with a laser
- matrix molecules absorb energy causing the matrix and sample to be volatised.
- Sample molecules pick up protons from matrix • an electric field accelerates ions
Matrix-Assisted Laser Desorption Ionisation
- high m/z values obtained (normal mass analysers unsuitable) so Time of Flight (TOF) analysis used
- all ions have the same kinetic energy (KE = 1⁄2mv2), so heavier ions move more slowly through a fixed distance between source and detector
• production of primarily singly charged ions means that there is one peak per component.
Accurate Mass Determination of Proteins
- No other technique can give a more accurate measurement of a protein’s molecular weight.
- Accuracy of 0.01% (ie. 1 in 10000 Da) can be achieved with standard instrumentation.
- We can check if a sequence is correct
- can distinguish between point mutations
• Useful for quality control of recombinant proteins in biotechnology.
Identifying a Protein by Mass Spectrometry
Used for the rapid identification of a protein of known sequence.
Used extensively in proteomics – analysis of proteins being expressed by a cell in a particular state or environment.
Identifying a Protein by Mass Spectrometry Methodology
Most proteins are too large for direct MS analysis and so are broken down into smaller pieces using an enzyme or chemical.
A wide range of residue specific enzymatic and chemical methods exist and more than one digest is performed.
Each reaction will give a different pattern of peptides
- The products are analysed by MS
ESMS coupled to HPLC or directly by MALDI
Compare the masses of the peptide fragments with those predicted from a genome sequence.
The peptide fragment patterns are likely to be unique for each protein .
Generation of Peptides from Proteins for Mass Spectrometric Analysis
Protease Enzymes
Trypsin
Chymotrypsin V8 Protease
0
Cleavage Site Lys
N – Xm – –/– Xn – C Arg
Phe
N – Xm – Try –/– Xn –
C Tyr
N – Xm - Glu –/– Xn – C
Generation of Peptides from Proteins for Mass Spectrometric Analysis
Chemical Methods
Chemical Methods
Cyanogen Bromide (BrCN)
2-Nitro-5-thio- Benzoic Acid (NTCBA)
Cleavage Site
N – Xm – Met –/– Xn –
C
N – Xm –/– Cys – Xn – C
0
Post-Translational Modifications
Proteins can be modified in many different ways after synthesis on the ribosome. Mass spectrometry is an ideal way to identify of a protein carries these modifications and where.
Post-Translational Modifications Phosphorylation
Occurs reversibly on serine, threonine and tyrosine.
Phosphorylation is an extremely important modification – it controls a wide range of protein activities and protein-protein interactions.
Mass shift 80 Da
Post-Translational Modifications Lysine Acetylation
Occurs reversibly on histone proteins. Controls histone binding to DNA and hence DNA replication and RNA transcription.
Mass shift 42 Da
Post-Translational Modifications: Glycosylation 1. N-Linked Glycosylation
Large oligosaccharides are attached to asparagine residues through N-acetylglucosamine (GlcNAc).
Found on cell-surface proteins. Modulates cell-cell interactions, protein folding and protein targeting.
Mass shift 203 Da
Post-Translational Modifications: Glycosylation 2. Cell-surface O-linked Glycosylation
Oligosaccharides (smaller than N-linked) attached to serine or threonine through N-acetyl- galactosamine. Similar functions to N-linked.
Mass shift 203 Da
Post-Translational Modifications: Glycosylation 2. Reversible O-GlcNAc-ylation
Attached to serine or threonine residues that are also sites of phosphorylation. Occurs on nuclear and cytoplasmic proteins. Function is not yet fully understood, but it may be involved in regulating phosphorylation.
Mass shift 203
Da 0
Post-Translational Modifications: Lipidation
Many proteins are modified by lipids, which result in anchoring of the protein to a membrane. They often form part of signal transduction complexes.
Myristoylation
Attached to N-terminal glycine
Mass shift 210 Da
Post-Translational Modifications: Lipidation
Farnesylation
Attached to cysteine four residues from C-terminus, followed by removal of last three residues and formation of methyl ester.
+ C15H25 – H (205 – 1)
+ OCH3 Mass shift 235 Da – 3 aa (31)
Sequencing a Protein by Mass Spectrometry
With more advanced instrumentation, principally based on electrospray, it is possible to completely sequence a protein by mass spectrometry.
Fragment the protein (enzymatic or chemical proteolysis such that relatively small fragments are obtained)
Analyse by HPLC-ESMS-MS
The first mass analyser is used to select a proteolytic peptide fragment (the parent ion)
Sequencing a Protein by Mass Spectrometry
The parent peptide ion is the allowed to collide with gas molecules (which induces further fragmentation – Collision Induced Dissociation)
Daughter fragment ions are then analysed in the second mass analyser
Sequencing a Protein by Mass Spectrometry
The peptide is cleaved inside the second mass spectrometer giving specific fragments.
Sequencing a Protein by Mass Spectrometry
- Compare results from different proteolytic reactions. The overlapping sequences can be combined to give the complete sequence
Post-translational modifications may be localised by observation of a mass shift of the fragments containing the modification.
Sequencing a Protein by Mass Spectrometry - limitations
- In practice can only get ≤ 15 amino acids of the sequence
- In general, cannot distinguish between leucine and isoleucine
- Interpretation of data is difficult and time consuming; searchable databases alleviate this problem.
Protein ligand binding
Mass spectrometry is ideal for studying covalent binding of enzyme substrates or inhibitors
The sequencing methods discussed previously can be used to identify active site amino acids involved in ligand binding.
Non-covalent ligand binding can also survive the ionisation process.
Can be used to study binding of metal ions to proteins
Mass Spectrometry of DNA
Electrospray can be used to observe:
- Single stranded DNA
- Double stranded (duplex) DNA •Quadruplex DNA
Sequence of DNA can be confirmed
Coupled to HPLC, electrospray mass spec can be used to analyse complex mixtures of DNA.
Mass Spectrometry of DNA
Problems:
Affinity of DNA for Na+ ions
leads to complex spectra; can be difficult to interpret spectra
Use ammonium acetate as the buffer which ensure DNA remains in duplex form
Ion peaks of ssDNA observed instead of dsDNA
strands dissociate during analysis; cold-spray ionization mass spectrometry can be used – carried out at –50 - 15°C so that unstable ions reach mass analyser before decomposing
Electrospray vs MALDI
Electrospray vs MALDI
