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Mass spectrometry basics
General scheme of Proteomic analysis
Protein Mixture
Peptide mixture Proteins
Peptides
M S Data
Identification
Digestion Separation
Separation Digestion
MS Analysis
Data Reduction Algorithm
Information from Mass spectrometryDetermination of Molecular weight.
Accuracy of 0.01% of the total molecular weight.
Changes in mass can be detected e.g. substitution of one amino acid for another, or a post-translational modification.
Structural information can be achieved by fragmenting the sample and analyzing the products generated.
Uses of mass spectrometerUsed in industry and academia for both routine and research purposes. Brief summary of the major mass spectrometric applications:
•Biotechnology: the analysis of proteins, peptides, oligonucleotides
•Pharmaceutical: drug discovery, combinatorial chemistry, pharmacokinetics, drug metabolism
•Clinical: neonatal screening, hemoglobin analysis, drug testing •Environmental: PAHs, PCBs, water quality, food contamination
•Geological: oil composition
Mass spectrometry for biochemists
Accurate molecular weight measurements:
confirmation of sample, determination of purity of a sample, verifying amino acid substitutions, detection of post-translational modifications, calculating number of disulphide bridges .
Reaction monitoring:
to monitor enzyme reactions, chemical modification, protein digestion
Amino acid sequencing:
sequence confirmation, de novo characterisation of peptides, identification of proteins by database searching with a sequence “tag” from a proteolytic fragment
Oligonucleotide sequencing:
the characterization or quality control of oligonucleotides
Protein structure:
protein folding monitored by H/D exchange, protein-ligand complex formation under physiological conditions, macromolecular structure determination
MS divided into 3 fundamental parts
Mass spectrometer
Data system
Ionization sourcee.g. electrospray(ESI),Matrix assisted laserDesorption(MALDI)
AnalyserMass to charge,m/ze.g. quadrupole,Time of flight, magnet, FT-ICR
Detectore.g. photomultiplier,Microchannel plate,Electron multiplier
Types of Mass spectrometry
Working of Mass Spectrometry
Divided into three fundamental parts:Ionization source Analyzer Detector
Sample is introduced in the ionization source where they are ionized. ( It is easier to manipulate ions than neutral molecules).
Ions separated according to their mass to charge ratio in the analyzer. The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum.
The separated ions are detected and this signal sent to a data system where the m/z ratios are stored together with their relative abundance for presentation in the format of a m/z spectrum.
Sample introduction
IonizationMinimize collisions, interferences
Separatemasses
Count ionsCollect results
Mass spectrometry Introduction
Sample introductionThe sample can be inserted directly into the ionization source, or can undergo some
type of chromatography en route to the ionisation source.
Gas source (lighter elements)dual inlet - sample purified and measured with standard gas at identical conditions
precisions ~ ±0.005%continuous flow - sample volatized and purified (by EA or GC) and injected into
mass spec in He carrier gas, standards measured before and after,precisions ~ 0.005-0.01%
Solid source (heavier elements)TIMS - sample loaded onto Re filament, heated to ~1500°C, precisions ~0.001%laser ablation - sample surface sealed under vacuum, then sputtered with laser
precisions ~0.01%
Inductively coupled plasma (all elements)ICPMS - sample converted to liquid form,
converted to fine aerosol in nebulizer,injected into ~5000K plasma torch
Matrix-Assisted Laser Desorption/Ionization (MALDI)
Used for nonvolatile & high Molecular analytes
biopolymers and oligomers
proteins, peptides, oligonucleotides, oligosaccharides
synthetic polymers
Inorganic, such a fullerenes
environmental compounds
kerogens, coal tars, humic acids, fulvic acids
MALDI Principle
A laser pulse is used for excitation
UV lasers cause electronic excitation
IR lasers cause vibrational excitation
Matrix molecules transfer energy
100-50,000 x [A]
low analyte fragmentation
matrices are selectable
TOF-MS is used for analysis
Matrices
Solid
co-crystallization of analyte and matrix
occasionally frozen solvents
Liquid
IR or UV absorbing liquid containing analyte
Two phase
liquids with absorbing solid
Common Matrices
α-cyno-4-hydroxycinnamic acid
2.5-dihydroxybenzoic acid Sinapinic acid
3-amino-4-hydroxybenzoic acid
Fragmentation
MALDI usually gives ‘molecular ions’
most often as protonated molecules
Control of fragmentation
differences in sublimation temperature of matrices control thermal excitation
PA and IP of a matrix affect energy transfer involved with protonation and electron transfer
collisions
extraction through neutral plume
residual gas
Ionization in MALDI
Ionization is separated into two divisions
primary ion formation
initial ions formed during laser pulse
frequently matrix molecules
secondary ion formation
ions formed during subsequent reactions
may be matrix-matrix reactions or matrix-analyte reactions
Resulting analyte ions are usually
protonated
Cationized
radical cations
Overview of Mass Spectrometry
Mass Spectrum
Mass Analyzer
Ionization M+/FragmentationSample Molecule (M)
Protonation : M + H+ MH Cationization : M + Cat+ MCat+
Deprotonation: MH M- + H+
Electron Ejection: M M+. + e-Electron Capture: M + e- M-.
Mechanism of Ionization
Matrix Assisted Laser Desorption Ionization (MALDI) (F. Hillenkamp, M. Karas, R. C. Beavis, B. T. Chait, Anal. Chem., 1991, 63, 1193), deals well with thermolabile, non-volatile organic compounds for the analysis of proteins, peptides, glycoprotein, oligosaccharides, and oligonucleotides.
Measures masses within 0.01% of the molecular weight of the sample, at least up to ca. 40,000 Da.
Based on the bombardment of sample molecules with a laser light to bring about ionisation.
Sample is pre-mixed with absorbing matrix compound for the most reliable results, and a low concentration of sample to matrix works best.
Matrix transforms the laser energy into excitation energy for the sample, leads to sputtering of analyte and matrix ions from the surface of the mixture.
In this way energy transfer is efficient and also the analyte molecules are spared excessive direct energy that may otherwise cause decomposition.
Most commercially available MALDI mass spectrometers now have a pulsed nitrogen laser of wavelength 337 nm.
Matrix Assisted Laser Desorption Ionisation
"Somehow, a peak seems to have appeared." Tanaka reported at the weekly Monday team meeting on February 2nd, 1985, half a year after the project had started.
Six O'clock in the Evening on October 9th 2002 News arrived saying that Koichi Tanaka had won the Nobel Prize in Chemistry 2002 On October 9th, the Royal Swedish Academy of Sciences announced their decision to award the Nobel Prize in Chemistry 2002 to three people for their development of methods for identification and structure analyses of biological macromolecules. - Koichi Tanaka (at the time : Life Science Laboratory Assistant Manager of Shimadzu Corporation), Prof. John B. Fenn (Virginia Commonwealth University, USA) and Prof. Kurt Wuthrich (Swiss Federal Institute of Technology)
1. Sample is mixed in matrix and dried on target.
2.Target is introduced into high vacuum of MS.
3.Sample is irradiated with laser desorbing ions into the gas phase and the clock measuring the time of flight starts.
4.Ions are accelerated by an electric field to the same kinetic energy and they drift down the field free flight tube where they are separated in space
5.Ions strike the detector at different times depending on the mass to charge ratio of the ions
6.A data system controls all the parameters, acquires the signal vs. time and permits data processing
Schematic of a MALDI-TOF Experiment
Diagrammatic representation
Laser induced ionization
Sample probe
vacuum
optics
Deflection plates
Ultraviolet laser
Data analysisoscilloscope
amplifier
MALDI-TOF Schematic
An aliquot of this removed and mixed with a solution containing a vast excess of a matrix. sinapinic acid( protein analysis), ±-cyano-4-hydroxycinnamic acid(peptide analysis)An aliquot applied to the sample, allowed to dry prior to insertion into the high vacuum. laser is fired, the energy arriving at the sample/matrix surface optimized, and data accumulated as m/z spectrum
Tof analyzer separates ions according to their m/z ratios. The heavier ions are slower than the lighter ones.
Results in the generation of singly charged ions regardless of the molecular weight.
In +ve ionisation mode the protonated molecular ions (M+H+) are usually the dominant species. Positive ionisation is used in general for protein and peptide analyses.
In -ve ionisation mode the deprotonated molecular ions (M-H-) are usually the most abundant species. Negative ionisation can be used for the analysis of oligonucleotides and oligosaccharides.
Sample dissolved in an appropriate volatile solvent
Flow chart
For a particle of Mass = m and charge = z, accelerated through a potential V between plates distance d apart: How long t does it take to complete the trip? What is final speed v? What is the relationship between t (time-of flight) and M/z (mass to charge ratio)?
Theoretical Basis of TOF Separations
Theoretical Basis for TOF-MS
Charge = zAccelerating voltage = VMass = mVelocity = vDistance = dTOF = t
zV = ½ mv2
V = ½ mv2/z2V= mv2/zbut v = d/tm/z = [2V/d2]t2
t = (m/z)1/2(d2/V)1/2
Molecular Ion[Mṇ]⁺
Dimer[MṇMṃ]⁺
Trimer
Tetramer
Molecular weight Determination
Sample ionization methodsThe ionisation methods used for the majority of biochemical analyses are
Electrospray Ionisation (ESI)
Matrix Assisted Laser Desorption Ionisation (MALDI).
Other Ionisation methods include:
Atmospheric Pressure Chemical Ionisation (APCI)
Chemical Ionisation (CI)
Electron Impact (EI)
Fast Atom Bombardment (FAB)
Field Desorption / Field Ionisation (FD/FI)
Thermo spray Ionisation (TSP)
Ion source mass analyzer detector
Involves transfer of molecules into a vacuum without decomposing them.
Discovered- in the late 80's in the group of Prof. Fenn at Yale.
The intact transfer of large molecules from the liquid gas phase by an ion desorption mechanism, a direct emission of large molecules from liquid droplets.
Operate under atmospheric conditions.
Electrospray Ionization
Electrospray Ionization (ESI)
The sample solution is sprayed across a high potential difference (a few kilovolts) from a needle into an orifice in the interface.
Heat and gas flows are used to desolvate the ions existing in the sample solution.
Electrospray ionization can produce multiply charged ions with the number of charges tending to increase as the molecular weight increases.
The number of charges on a given ionic species must be determined by methods such as:
comparing two charge states that differ by one charge and solving simultaneous equations
looking for species that have the same charge but different adduct masses
examining the mass-to-charge ratios for resolved isotopic clusters
Electron Spray Ionization(ESI)(J. Fenn, J. Phys. Chem., 1984, 88, 4451)
Polar molecule analysis, molecules ranging from less than 100 Da to more than 1,000,000 Da in molecular weight.
ProcedureSample is dissolved in a polar, volatile solvent and pumped through a narrow, stainless steel capillary. High voltage applied to the tip of the capillary.Sample emerging from the tip is dispersed into an aerosol of highly charged droplets, aided by nebulising gas flowing around the outside of the capillary.Charged droplets diminish in size by solvent evaporation, assisted by a warm flow of nitrogen known as the drying gas which passes across the front of the ionisation source. Charged sample ions, free from solvent, are released from the droplets, pass through an orifice into an intermediate vacuum region, and from there into the analyzer of the mass spectrometer, under high vacuum.
Diagrammatic of Electronspray Ionization
Mechanism of Electronspray ionization
Original dropletContains + and – Ions; + predominant
+ + - +- + -- + +
- +
++ +
-+ + -
++
+
Solvent evaporatesField increases, andIons move towardsurface
As fieldincreases,ions areemitted fromdrop
In volatileresidue
The micro droplet shrinks due to solvent evaporation The resulting increase in charge density of the droplet, forces the charged analyte ion out of the solution before the droplet breaks up.
Sample introduction
Flow injection
LC/MS
Typical flow rates are less than 1 micro liter per minute up to about a milliliter per minute.
Benefits
Good for charged, polar or basic compounds
Permits the detection of high-mass compounds at mass-to-charge ratios that are easily determined by most mass spectrometers (m/z typically less than 2000 to 3000).
Best method for analyzing multiply charged compounds.
Very low chemical background leads to excellent detection limits.
Can control presence or absence of fragmentation by controlling the interface lens potentials.
Compatible with MS/MS methods.
Limitations
Multiply charged species require interpretation and mathematical transformation (can be difficult sometimes).
Complementary to APCI. Not good for uncharged, non-basic, low-polarity compounds (e.g. steroids).
Very sensitive to contaminants such as alkali metals or basic compounds.
Relatively low ion currents
Relatively complex hardware compared to other ion sources
Mass range
Low-high Typically less than 200,000 Da.
In positive ionization mode, a trace of formic acid is often added to aid protonation of the sample molecules.
In negative ionization mode a trace of ammonia solution or a volatile amine is added to aid deprotonation of the sample molecules.
Proteins and peptides are usually analyzed under positive ionisation conditions and saccharides and oligonucleotides under negative ionisation conditions.
In all cases, the m/z scale must be calibrated by analyzing a standard sample.
Positive or Negative Ionizsation? If the sample has functional groups that readily accept a proton (H+) then positive
ion detection is used e.g. amines R-NH2 + H+ ® R-NH3+ as in proteins, peptides
If the sample has functional groups that readily lose a proton then negative ion detection is used e.g. carboxylic acids R-CO2H ® R-CO2– and alcohols R-OH ® R-O– as in saccharides, oligonucleotides
Positive and negative Ion mode
In ESI, samples (M) up to 1200 Da give rise to singly charged molecular-related ions, usually protonated molecular ions of the formula (M+H)+ in positive ionisation mode and deprotonated molecular ions of the formula (M-H)- in negative ionisation mode.
Samples (M) with molecular weights > 1200 Da give rise to multiply charged molecular-related ions such as (M+nH)n+ in positive ionisation mode and (M-nH)n- in negative ionisation mode.
Proteins have many suitable sites for protonation as all of the backbone amide nitrogen atoms could be protonated theoretically, as well as certain amino acid side chains such as lysine and arginine which contain primary amine functionalities.
m/z = (MW + nH+)/ n
where m/z = the mass-to-charge ratio marked on the abscissa of the spectrum;
MW = the molecular weight of the sample n = the integer number of charges on the ions H = the mass of a proton = 1.008 Da.
Expression of m/z value
Leucine enkephalin
TEST0132(1.679)cm(3:34)
Platform II, BMB, University of Leeds
ESI-MS analysis of Leucine enkephalinCalculated MW.555.2 DaMeasured MW.555.1Da
4 Oct 199910:12:26
Scan ES+2.87 e5
M/Z Spectrum in positive ionization mode
The m/z spectrum also contains ions at m/z 578.1, some 23 Da higher than the expected molecular weight. These can be identified as the sodium adduct ions, (M+Na)+, and are quite common in electrospray ionization.
Interpretation
Electrospray ionization is known as a “soft” ionization method as the sample is ionised by the addition or removal of a proton, with very little extra energy remaining to cause fragmentation of the sample ions
By raising the voltage applied to the sampling cone, extra energy is supplied to the sample ions which can then fragment. The m/z spectrum then has extra peaks corresponding to sample fragment ions which can help in the structural elucidation of the sample.
Known as “cone voltage” or “in-source” fragmentation and although it can provide useful information it must be remembered that it is not specific so if there are a number of components in a sample, all will fragment to give rise to an extremely complicated spectrum.
Advantages of Multiple Charging
Can use instruments with lower maximum m/z (i.e., Quadrupoles, ion traps, FTMS)
For FTMS, the resolution is better at lower m/z values, therefore, ESI helps one obtain better resolution at higher m/z values.
Multiply charge ions tend to fragment easier then singly charge ions.
M/Z value expression
Platform II, BMB, University of Leeds
Hen egg Lysozyme
LYSO1A 1(1.392)Sm(SG, 2X1.00): Sb(10.10.00)
25 Jan 2000 10:10:37
ScanES+ 2.16e6
m/z = (MW + nH+)
n where
m/z = the mass-to-charge ratio marked on the abscissa of the spectrum;MW = the molecular weight of the samplen = the integer number of charges on the ionsH = the mass of a proton = 1.008 Da.
M/Z value expression
1431.6 = (MW + nH+) and 1301.4 = (MW + (n+1)H+) n (n+1)
These simultaneous equations can be rearranged to exclude the MW term:
n(1431.6) –nH+ = (n+1)1301.4 – (n+1)H+
and so n(1431.6) = n(1301.4) +1301.4 – H+
therefore: n(1431.6-1301.4)= 1301.4 – H+
and: n= (1301.4 - H+)
(1431.6 – 1301.4) hence the number of charges on
the ions at m/z 1431.6 = 1300.4 = 10. 130.2
1431.6 = (MW + nH+)n
gives 1431.6 x 10 = MW + (10 x
1.008) and so MW = 14,316 – 10.08 therefore MW = 14,305.9 Da
Peptide Mass Fingerprinting
peptides
Peptide mass fingerprint Peptide fragments
Peptide mass search MS/MS search
Schematic representation of MALDI-TOF mass spectrometer
MALDI ionization process MALDI-TOF operating in linear mode
MALDI-TOF instrument equipped with a reflection
Peptide Mass Fingerprinting
Effect of Mass accuracy and Mass Tolerance on peptide Mass Fingerprinting search result
Search m/z Mass tolerance #Hits
1529 1 478
1529.7 0.1 164
1529.73 0.01 25
1529.734 0.001 4
1529.7348 0.0001 2
Searches were done with the MS-FIT program
Effect of Multiple Peptide Masses on Protein Identification
Search m/z Mass tolerance #Hits
1529.73 0.1 204
1529.73
1529.7O 0.1 7
1529.73
1252.7O1833.88 0.1 1
Searches were done with the MS-FIT program
The Actual Peptide M/Z values are 1529.7348,1252.7074,1833.8845
Protein matches for peptide Mass Fingerprinting of m/z 1529.73
Peptide sequence identification Matched m/z
IGGHGAEYGAEALER Mouse Hb alpha 1529.7348
VGAHAGEYGAEALER Human Hb alpha 1529.7348
MGTGWEGMYRTLK Mouse lens epithelialCell protein LEP503
1529.7245
MADEEKLPPGWEK Human PINI-like protein
1529.7310
DTQTSITDSSAIYK Mouse signal recognition particle
1529.7335
NDSSPNPVYQPPSK Mouse peroxisome assembly factor-1
1529.7236
MNLSLNDAYDFVK Human dual specificity protein phosphatase 7
1529.7310
Interpretation of PMFExercise for participants
+
Carbamidomethylation 57 Da
Total 2188 Da
+
N-terminal Peptide Identified N-terminal Peptide Identified
Insilico digested N-terminal peptide for FILGRASTRIM
MTPLGPASSLPQFLIKCLEN-terminal peptide sequence
m/z=1095
MTPLGPASSLPQFLIKCLEN-terminal peptide sequence
m/z=1095
MTPLGPASSLPQFLIKCLEAbsence of N-terminal sequence
m/z=1095
MTPLGPASSLPQFLIKCLEAbsence of N-terminal sequence
m/z=1095
MTPLGPASSLPQFLIKCLEN-terminal peptide sequence
m/z=1095
MTPLGPASSLPQFLIKCLEN-terminal peptide sequence
m/z=1095
Schematic of Triple-Quad MS
Mass Changes from Post translational Modification
Mass Change Modification -2.0 Disulfide bond formation+14.0 Methylation+16.0 Hydroxylation+28.0 Formylation+30.0 Nitrosylation+42.0 Acetylation+80.0 Sulfation+80.1 Phosphorylation+180 Mono-glycosylation+204.4 Farnesylation+210.4 Myristoylation