Mass Spectrometry 101 - PowerPoint PPT Presentation

Loading...

PPT – Mass Spectrometry 101 PowerPoint presentation | free to download - id: 547978-MzQ0M



Loading


The Adobe Flash plugin is needed to view this content

Get the plugin now

View by Category
About This Presentation
Title:

Mass Spectrometry 101

Description:

Mass Spectrometry 101 An Introductory Lecture On Mass Spectrometry Fundamentals Presented to the Sandler Mass Spectrometry Users Group University of California San ... – PowerPoint PPT presentation

Number of Views:965
Avg rating:3.0/5.0
Slides: 59
Provided by: Appli4
Learn more at: http://docencia.izt.uam.mx
Category:

less

Write a Comment
User Comments (0)
Transcript and Presenter's Notes

Title: Mass Spectrometry 101


1
Mass Spectrometry 101 An Introductory Lecture On
Mass Spectrometry Fundamentals
Presented to the Sandler Mass Spectrometry Users
Group University of California San
Francisco April 11, 2003
http//www.google.com/url?satsourcewebcd12ve
d0CCIQFjABOAourlhttp3A2F2Fwww.msg.ucsf.edu2
Fagard2Fmaldi2FIntrotoMS.pptrctjqmass20spec
trometry20ppteia9Q8TY6fJMXYgAf-mqn_CAusgAFQjC
NEQD59F-CzruumQAKfj5xIO7xLH0Qsig2-L7h-VZMUVu_Cz1
EoTNTnQcadrja
2
What does a mass spectrometer do? 1. It measures
mass better than any other technique. 2. It can
give information about chemical structures. What
are mass measurements good for? To identify,
verify, and quantitate metabolites, recombinant
proteins, proteins isolated from natural sources,
oligonucleotides, drug candidates, peptides,
synthetic organic chemicals, polymers
3
Applications of Mass Spectrometry
Pharmaceutical analysis Bioavailability
studies Drug metabolism studies,
pharmacokinetics Characterization of potential
drugs Drug degradation product analysis Screening
of drug candidates Identifying drug
targets Biomolecule characterization Proteins and
peptides Oligonucleotides Environmental
analysis Pesticides on foods Soil and groundwater
contamination Forensic analysis/clinical
4
How does a mass spectrometer work?
Sample
Mass analyzer separates ions
Mass spectrumpresents information
5
Mass Spectrometer Block Diagram
High Vacuum System
Ion source
Mass Analyzer
Data System
Inlet
Detector
6
Mass Spectrometer Block Diagram
Turbo molecular pumps
High Vacuum System
Ion source
Mass Analyzer
Data System
Inlet
Detector
7
Sample Introduction
High Vacuum System
Ion Source
Mass Analyzer
Data System
Inlet
Detector
HPLC Flow injection Sample plate
8
Ion Source
High Vacuum System
Ion Source
Mass Analyzer
Data System
Inlet
Detector
MALDI ESI FAB LSIMS EI CI
9
Electrospray ionization
10
MALDI Matrix Assisted Laser Desorption Ionization
Sample plate
Laser
hn
1. Sample is mixed with matrix (X) and dried on
plate. 2. Laser flash ionizes matrix
molecules. 3. Sample molecules (M) are ionized by
proton transfer XH M ? MH X.
MH
Grid (0 V)
/- 20 kV
11
Mass Analyzer
High Vacuum System
Ion source
Mass Analyzer
Data System
Inlet
Detector
Time of flight (TOF) Quadrupole Ion Trap Magnetic
Sector FTMS
12
Mass analyzers separate ions based on
their mass-to-charge ratio (m/z)
  • Operate under high vacuum (keeps ions from
    bumping into gas molecules)
  • Actually measure mass-to-charge ratio of ions
    (m/z)
  • Key specifications are resolution, mass
    measurement accuracy, and sensitivity.
  • Several kinds exist for bioanalysis, quadrupole,
    time-of-flight and ion traps are most used.

13
Quadrupole Mass Analyzer
Uses a combination of RF and DC voltages to
operate as a mass filter.
  • Has four parallel metal rods.
  • Lets one mass pass through at a time.
  • Can scan through all masses or sit at one fixed
    mass.

14
Quadrupoles have variable ion transmission modes
15
Time-of-flight (TOF) Mass Analyzer
Source
Drift region (flight tube)


detector


V
  • Ions are formed in pulses.
  • The drift region is field free.
  • Measures the time for ions to reach the
    detector.
  • Small ions reach the detector before large ones.

16
Ion Trap Mass Analyzer
Top View
Cut away side view
17
Detector
High Vacuum System
Ion source
Mass Analyzer
Data System
Inlet
Detector
Microchannel Plate Electron Multiplier Hybrid
with photomultiplier
18
Ions are detected with a microchannel plate
19
Data System
High Vacuum System
Ion source
Mass Analyzer
Data System
Inlet
Detector
PC Sun SPARK Station DEC Station
20
The mass spectrum shows the results
MALDI TOF spectrum of IgG
MH
Relative Abundance
(M2H)2
(M3H)3
50000
100000
150000
200000
Mass (m/z)
21
ESI Spectrum of Trypsinogen (MW 23983)
M 15 H
1599.8
M 16 H
M 14 H
1499.9
1714.1
M 13 H
1845.9
1411.9
1999.6
2181.6
m/z
Mass-to-charge ratio
22
  • How do mass spectrometers get their names?
  • Types of ion sources
  • Electrospray (ESI)
  • Matrix Assisted Laser Desorption Ionization
    (MALDI)
  • Types of mass analyzers
  • Quadrupole (Quad, Q)
  • Ion Trap
  • Time-of-Flight (TOF)
  • Either source type can work with either analyzer
    type MALDI-TOF, ESI-Quad.
  • Analyzers can be combined to create hybrid
    instruments. ESI-QQQ, MALDI QQ TOF, Q Trap

23
Voyager-DE STR MALDI TOF
Sample
Linear
Extraction
plate
Reflector
detector
grids
Timed ion
detector
selector
Reflector
Laser
Camera
Pumping
Pumping
24
QSTARTM ESI QQ TOF or MALDI QQ TOF
Sample
Q0
Q1
Q2
Effective Flight Path 2.5 m
Ion Mirror (reflector)
25
QTRAP Linear Ion Trap on a Triple Quadrupole
A new type of instrument.
Q0
Q1
Q2
Q3
Exit
linear ion trap
26
Summary acquiring a mass spectrum
Ionization
Mass Sorting (filtering)
Detection
Ion Source
Ion Detector
Mass Analyzer
Form ions (charged molecules)
Sort Ions by Mass (m/z)
Detect ions
100
75
Inlet
Solid Liquid Vapor
50
25
0
1330
1340
1350
Mass Spectrum
27
How is mass defined?
Assigning numerical value to the intrinsic
property of mass is based on using carbon-12,
12C, as a reference point. One unit of mass is
defined as a Dalton (Da). One Dalton is defined
as 1/12 the mass of a single carbon-12
atom. Thus, one 12C atom has a mass of 12.0000
Da.
28
Isotopes
Most elements have more than one stable
isotope. For example, most carbon atoms have a
mass of 12 Da, but in nature, 1.1 of C atoms
have an extra neutron, making their mass 13 Da.
Why do we care? Mass spectrometers can see
isotope peaks if their resolution is high
enough. If an MS instrument has resolution high
enough to resolve these isotopes, better mass
accuracy is achieved.
29
Stable isotopes of most abundant elements of
peptides
30
Mass spectrum of peptide with 94 C-atoms (19
amino acid residues)
Monoisotopic mass
No 13C atoms (all 12C)
One 13C atom
Two 13C atoms
31
Isotope pattern for a larger peptide (207 C-atoms)
32
Mass spectrum of insulin
2 x 13C
13C
12C 5730.61
Insulin has 257 C-atoms. Above this mass, the
monoisotopic peak is too small to be very useful,
and the average mass is usually used.
33
Monoisotopic mass
When the isotopes are clearly resolved the
monoisotopic mass is used as it is the most
accurate measurement.
34
Average mass
Average mass corresponds to the centroid of the
unresolved peak cluster
When the isotopes are not resolved, the centroid
of the envelope corresponds to the weighted
average of all the the isotope peaks in the
cluster, which is the same as the average or
chemical mass.
35
What if the resolution is not so good? At lower
resolution, the mass measured is the average mass.
Better resolution
Poorer resolution
6130
6140
6150
6160
6170
Mass
36
How is mass resolution calculated?
M
R M/DM
FWHM DM
37
Mass measurement accuracy depends on resolution
High resolution means better mass accuracy

15 ppm error
24 ppm error
55 ppm error
38
How do we achieve superior mass resolution?
Reflector TOF Mass Analyzer
Delayed Extraction on a MALDI source
39
Important performance factors
Mass accuracy How accurate is the mass
measurement? Resolution How well separated are
the peaks from each other? Sensitivity How
small an amount can be analyzed?
40
What is MSMS?
MS/MS means using two mass analyzers (combined in
one instrument) to select an analyte (ion) from a
mixture, then generate fragments from it to give
structural information.
Mixture of ions
Single ion
Fragments
Ion source
MS-2
MS-1
41
What is MS/MS?
1 peptide selected for MS/MS
Peptide mixture


MS/MS



The masses of all the pieces give an MS/MS
spectrum
Have only masses to start
42
Interpretation of an MSMS spectrum to derive
structural information is analogous to solving a
puzzle





Use the fragment ion masses as specific pieces of
the puzzle to help piece the intact molecule back
together
43
Cleavages Observed in MS/MS of Peptides
zn-i
vn-i
wn-i
-HN--CH--CO--NH--CH--CO--NH-
Ri
CH-R
R
ci
di1
44
EGlu GGly SSer FPhe NAsn PPro VVal AAla R
Arg
Peptide Fragmentation
gt
E
G
S
F
F
G
E
E
N
P
N
V
A
R
175.10 246.14 345.21 459.25 556.30 670.35 799.39 9
28.43 985.45 1132.52 1279.59 1366.62 1423.64 1552.
69



45
Protein Identification
1. Peptide Mass Finger Printing (PMF) from MS
data 2. Database search using fragment ion
masses from MS/MS data 3. Sequence
Tags from MS/MS data
46
PROBLEM
  • Biologist
  • What protein was isolated?
  • Bank President
  • Who robbed the bank?

47
GATHER EVIDENCE
  • Mass Spectrometrist
  • 1. Interview biologist who isolated the protein
  • 2. Cleave protein to obtain peptide mixture
  • 3. Analyze peptide mixture by MS to obtain
    peptide molecular masses!
  • Police Officer
  • 1. Interview witnesses
  • 2. Dust for fingerprints

enzyme
48
DATABASE SEARCH
  • Mass Spectrometrist
  • Approx. molecular weight 30,000
  • Origin bovine liver
  • Peptide mass list from MS analysis 975.4832,
    1112.5368, 632.3147, 803.4134, 764.3892
  • Police Officer
  • Height 57
  • Weight 160 lbs
  • Gender male
  • Age 35-40
  • Fingerprints

search
search
DATABASE OF KNOWN FELONS
PEPTIDE MASS DATABASE OF KNOWN PROTEINS
49
DATABASE SEARCH RESULTS
  • Police Officer
  • Identifies the robber
  • Anthony J. Felon
  • Mass Spectrometrist
  • Identifies the protein
  • bovine carbonic anhydrase

50
(No Transcript)
51
Mass accuracy tolerance 15 ppm This means that
the mass is within 0.015 Da at m/z 1000
52
(No Transcript)
53
(No Transcript)
54
MS/MS spectrum for tryptic peptide MH 1025.5,
EAFQLFDR, from Spot A. An MS-Tag search using
the fragment ions from this spectrum confirmed
the identity of Spot A as myosin light chain.
55
Sequence Tags from Peptide Fragmentation by MS/MS
One sequence tag includes four components
  • peptide molecular weight (MW)
  • partial sequence (region 2)
  • molecular wt before partial sequence (region 1)
  • molecular wts after partial sequence (region 3)

1546.11
56
Sequence TAG Example from MS/MS Spectrum Peptide
MW 1345.70
57
Sequence Tag search identifies 1 hit (carbonic
anhydrase)
58
Acknowledgements
We thank the Applied Biosystems Mass Spectrometry
Applications Laboratory for allowing the use of
some of their slides for this presentation.
About PowerShow.com