What does a mass spectrometer do?

Mass Spectrometry 101 1. It measures mass (m/z) better than any other
technique.
2. It can give information about chemical structures.
Hackert - CH 370 / 387D What are mass measurements good for?
To identify:
metabolites, synthetic organic chemicals
Based in part on Lecture Notes from peptides, proteins, recombinant proteins,
“An Introductory Lecture On Mass Spectrometry Fundamentals” oligonucleotides, polymers
Presented to the Sandler Mass Spectrometry Users’ Group,
University of California San Francisco, and
drug candidates,
“Fundamentals of Mass Spectrometry – Based Proteomics” complexes
by Doug Sheeley – Division of Biomedical Technology, National ,
Center for Research Resources

Summary: acquiring a mass spectrum

Who uses mass measurements?
Ionization Mass Sorting (filtering) Detection
Pharmaceutical analysis
Bioavailability studies Ion Ion
Source Mass Analyzer Detector
Drug metabolism studies, pharmacokinetics
Characterization of potential drugs Form ions
Sort Ions by Mass (m/z) Detect ions
(charged molecules)
Drug degradation product analysis Time of flight (TOF) Microchannel Plate
Screening of drug candidates Quadrupole Electron Multiplier
Identifying drug targets Ion Trap Hybrid / photomultiplier
Biomolecule characterization Magnetic Sector
Inlet • Solid
Proteins and peptides • Liquid
FTMS
100
• Vapor
Oligonucleotides 75

50

Environmental analysis MALDI

25

0
1330 1340 1350

Pesticides on foods ESI Mass Spectrum

Soil and groundwater contamination FAB
EI
Forensic analysis/clinical
CI

1
 Mass Spectrometry – Focus on Proteomics Mass Spectrometry

Source: produces charged particles (ions)
• Electron Impact (EI) - Hard (fragments) / 1000 Da
• Chemical Ionization (CI) – (methane / isobutane / ammonia)
• Fast Atom Bombardment (FAB) – 6keV xenon atoms
• Electrospray Ionization (ESI) - Soft / 200kDa
Introductory Example: mass spectrum of water
• Electron Impact (EI) - Hard (fragments) / 1000 Da
H2O + fast electron Æ [H2O]+ + 2 electrons
+ fragments ([OH]+, O+, H+)
Fragmentation pattern
[H2O]+ 18

Relative Abundance
Molecular Ion
• Matrix-Assisted Laser Desorption Ionization - Soft / 500kDa
[OH]+ 17 (“fingerprint”)
O+ 16
H+ 1
1 16 17 18
Mass-to-charge ratio

How is mass defined?

Isotopes
Assigning numerical value to the intrinsic property
of “mass” is based on using carbon-12, 12C, as a +Most elements have more than one stable isotope.
reference point. 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.
One unit of mass is defined as a Dalton (Da).
+Why do we care?
One Dalton is defined as 1/12 the mass of a Mass spectrometers can “see” isotope peaks if their resolution
is high enough.
single carbon-12 atom.
If an MS instrument has resolution high enough to resolve these
isotopes, better mass accuracy is achieved.
Thus, one 12C atom has a mass of 12.0000 Da.

2
Stable isotopes of most abundant elements of Monoisotopic mass
peptides
Monoisotopic mass
Element Mass Abundance corresponds to
H 1.0078 99.985% lowest mass peak
2.0141 0.015
C 12.0000 98.89
13.0034 1.11
N 14.0031 99.64
15.0001 0.36
O 15.9949 99.76
16.9991 0.04 When the isotopes are clearly resolved the monoisotopic mass
17.9992 0.20 is used as it is the most accurate measurement.

Mass spectrum of peptide with 94 C-atoms Mass spectrum of insulin

(19 amino acid residues)
2 x 13C
“Monoisotopic mass”
No 13C atoms (all 12C)
1981.84 13C

1982.84 One 13C atom

12C : 5730.61
1983.84
Two 13C atoms

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.

3
 Average mass What if the resolution is not so good?
Average mass corresponds At lower resolution, the mass measured is the average mass.
to the centroid of the
unresolved peak cluster
Better
Poorer
resolution
resolution

When the isotopes are not resolved, the centroid of the envelope 6130 6140 6150 6160 6170
Mass
corresponds to the weighted average of all the the isotope peaks in
the cluster, which is the same as the average or chemical mass.

ISO:CH3
How is mass resolution calculated?
M15.0229
100
100
Mass measurement accuracy depends on resolution
90

80
High resolution means better mass accuracy
70
R = M/ΔM Resolution =18100
8000
15 ppm error
60
% Intensity

FWHM = ΔM 6000

Counts
50 Resolution = 14200
24 ppm error
4000
40

Resolution = 4500
30 2000
55 ppm error
20 0

2840 2845 2850 2855

10
Mass (m/z)

0
15.01500 15.01820 15.02140 15.02460 15.02780 15.03100
Mass (m/z)

4
 How do mass spectrometers get their names?
ESI QQ TOF or MALDI QQ TOF
Types of ion sources:
• Electrospray (ESI) - Soft / 200kDa
Sample • Matrix Assisted Laser Desorption Ionization (MALDI) ~ 500kDa

Q0 Q1 Types of mass analyzers:

Q2
• Quadrupole (Quad, Q)
• Time-of-Flight (TOF)
• Ion Trap
Effective Flight
Path = 2.5 m -Either source type can work with either analyzer type: “MALDI-
TOF,” “ESI-Quad.”
Ion Mirror
(reflector)
-Analyzers can be combined to create “hybrid” instruments. ESI-
QQQ, MALDI QQ TOF, Q Trap

Ion Sources make ions from sample molecules

(Ions are easier to detect than neutral molecules.)
Electrospray ionization:

Sample Inlet Nozzle Partial

Pressure = 1 atm vacuum
Inner tube diam. = 100 um (Lower Voltage)

N2 MH+
+
++++ + ++ + +
++ + +
++
++ + +
Sample in solution
++
+
+ ++ +
+
++
+ +
+ + MH2+
++ ++ + ++ +
N2 gas ++ + +

MH3+

High voltage applied Charged droplets

to metal sheath (~4 kV)

Very gentle / solvent evaporates / many, multiple charges (H+)

5
 MALDI (Matrix Assisted Laser Desorption Ionization)
ESI Spectrum of Trypsinogen (MW 23,983)
“Nuke It”

1599.8 M + 15 H+
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

MALDI: Matrix Assisted Laser Desorption Ionization

Sample plate
Laser
hν
Sinapinic acid

1. Sample is mixed with matrix (X)

MH+ and dried on plate.
Cyano-4-hydroxy-trans –cinnamic acid
2. Laser flash ionizes matrix
molecules.
3. Sample molecules (M) are
ionized by proton transfer:
XH+ + M Æ MH+ + X. 2,5-Dihydroxybenzoic acid

+/- 20 kV Grid (0 V)

6
 Summary: acquiring a mass spectrum
Mass analyzers separate ions based on their
Ionization Mass Sorting (filtering) Detection
mass-to-charge ratio (m/z)
Ion Ion
Source Mass Analyzer Detector ¤ Operate under high vacuum (keeps ions from bumping
Form ions into gas molecules)
Sort Ions by Mass (m/z) Detect ions
(charged molecules)
Time of flight (TOF) Microchannel Plate
¤ Actually measure mass-to-charge ratio of ions (m/z)
Quadrupole Electron Multiplier ¤ Key specifications are resolution, mass measurement
Ion Trap Hybrid / photomultiplier
Magnetic Sector accuracy, and sensitivity.
Inlet • Solid
FTMS
•
•
Liquid
Vapor
100 ¤ Several kinds exist: for bioanalysis, quadrupole, time-of-
75

50
flight and ion traps are most used.
25

MALDI 0
1330 1340 1350

ESI Mass Spectrum

FAB
EI
CI

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.

7
Quadrupoles have variable ion transmission modes Time-of-flight (TOF) Mass Analyzer
Source Drift region (flight tube)
m2 m1

m4 m3 m2 m1 +
+

detector
m4 m3

mass scanning mode + +

m2 m1 V

m4 m3
m2 m2 m2 m2 • Ions are formed in pulses.
• The drift region is field free.

single mass transmission mode • Measures the time for ions to reach the detector.
• Small ions reach the detector before large ones.

The mass spectrum shows the results

MALDI TOF spectrum of IgG

40000 MH+

Relative Abundance
30000

(M+2H)2+
20000

10000
(M+3H)3+

50000 100000 150000 200000

Mass (m/z)

8
 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 Single Fragments

ions ion

Ion
MS-1 MS-2
source

9
Cleavages Observed in MS/MS of Peptides
yn-i
zn-i
low energy
xn-i vn-i wn-i

-HN--CH--CO--NH--CH--CO--NH-
Ri CH-R’
ai
bi
R” high energy

ci
di+1

What is MS/MS?

Peptide 1 peptide
selected for
mixture MS/MS

+
MS/MS +
+
+ +

Have only masses The masses of all

to start the pieces give an
MS/MS spectrum

10
Interpretation of an MSMS spectrum to derive E=Glu
G=Gly
structural information is analogous to solving a Peptide Fragmentation S=Ser
F=Phe
puzzle N=Asn
P=Pro
V=Val
A=Ala
=> E G S F F G E E N P N V A R R=Arg
+
175.10
+
+
246.14 =
345.21
+ + 459.25
556.30
670.35 =
799.39
928.43
985.45
Use the fragment ion masses as specific pieces of 1132.52
1279.59
the puzzle to help piece the intact molecule back 1366.62
together 1423.64 =
1552.69

11
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