2. General scheme of Proteomic analysis
Protein Mixture
Digestion Separation
Peptide mixture Proteins
Separation Digestion
Peptides
MS Analysis
M S Data
Data Reduction Algorithm
Identification
3. Information from Mass spectrometry
Determination 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.
4. Uses of mass spectrometer
Used 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
5. 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
6. MS divided into 3 fundamental parts
Mass spectrometer
Data system
Ionization source Analyser Detector
e.g. electrospray(ESI), Mass to charge,m/z e.g. photomultiplier,
Matrix assisted laser e.g. quadrupole, Microchannel plate,
Desorption(MALDI) Time of flight, Electron multiplier
magnet, FT-ICR
8. 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.
10. Sample introduction
The 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
11. 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
12. 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
15. 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
16. 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
17. Overview of Mass Spectrometry
Sample Molecule (M) Ionization M+/Fragmentation
Mass Analyzer
Mass Spectrum
Protonation : M + H+ MH
Cationization : M + Cat+ MCat+
Mechanism of Ionization
Deprotonation: MH M - + H+
Electron Ejection: M M+. + e-
Electron Capture: M + e- M-.
18. Matrix Assisted Laser Desorption Ionisation
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.
19. "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)
20. Schematic of a MALDI-TOF Experiment
4.Ions are accelerated by an electric
field to the same kinetic energy and
1. Sample is they drift down the field free flight
mixed in tube where they are separated in
matrix and space
dried on
target.
5.Ions strike the
2.Target is introduced detector at different
into high vacuum of times depending on
MS. the mass to charge
ratio of the ions
3.Sample is irradiated
with laser desorbing
ions into the gas phase
and the clock
measuring the time of 6.A data system controls all
flight starts. the parameters, acquires the
signal vs. time and permits
data processing
24. Flow chart
Sample dissolved in an appropriate volatile solvent
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.
25. Theoretical Basis of TOF Separations
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)?
26. Theoretical Basis for TOF-MS
Charge = z zV = ½ mv2
Accelerating voltage = V = ½ mv2/z
V 2V= mv2/z
Mass = m but v = d/t
Velocity = v m/z = [2V/d2]t2
Distance = d t = (m/z)1/2(d2/V)1/2
TOF = t
30. Electrospray Ionization
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.
31. 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
32. 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.
Procedure
Sample 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.
34. Mechanism of Electronspray ionization
+
+ + - + + +
- + - +
- + + + + ++-
- + -
In volatile
Solvent evaporates As field
Original droplet
Field increases, and increases, residue
Contains + and –
Ions move toward ions are
Ions; +
surface emitted from
predominant
drop
35. 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.
37. 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.
38. 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.
39. Positive and negative Ion mode
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
40. 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.
41. Expression of m/z value
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.
42. M/Z Spectrum in positive ionization mode
Leucine enkephalin Platform II, BMB, University of Leeds 4 Oct 199910:12:26
TEST0132(1.679)cm(3:34) Scan ES+
2.87 e5
ESI-MS analysis of Leucine enkephalin
Calculated MW.555.2 Da
Measured MW.555.1Da
43. Interpretation
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.
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.
44. 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.
45. M/Z value expression
LYSO1A 1(1.392)Sm(SG, 2X1.00): Sb(10.10.00) Platform II, BMB, University of Leeds
25 Jan 2000 10:10:37
Hen egg
Lysozyme ScanES+ 2.16e6
46. M/Z value expression
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.
47. 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
48. 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
51. Schematic representation of MALDI-
TOF mass spectrometer
MALDI ionization process MALDI-TOF operating in linear mode
MALDI-TOF
instrument
equipped with a
reflection
53. 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
54. 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
1252.7O
1833.88 0.1 1
Searches were done with the MS-FIT program
The Actual Peptide M/Z values are 1529.7348,1252.7074,1833.8845
55. 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 epithelial 1529.7245
Cell protein LEP503
MADEEKLPPGWEK Human PINI-like 1529.7310
protein
DTQTSITDSSAIYK Mouse signal 1529.7335
recognition particle
NDSSPNPVYQPPSK Mouse peroxisome 1529.7236
assembly factor-1
MNLSLNDAYDFVK Human dual 1529.7310
specificity protein
phosphatase 7
