The “LC” in LC/MS The “MS” in LC/MS

• High Performance Liquid Chromatography • Mass Spectrometry

– Separates compounds based on their chemical – A process in which ions are generated and analyzed
characteristics (e.g. polar, nonpolar, acidic, basic, according to their mass-to-charge ratio (m/z) and in
etc.) which the number of ions is determined electrically.

The “/” – The Transition in LC/MS Typical LC/MS System Progression

ANALYSER
ION DETECTOR
• Fact: Compounds in liquid phase eluting from the +
+
HPLC at atmospheric pressure SOURCE
+ +
+
+ +
+
+
+
• Fact: Mass spectrometer “accepts” only gas phase ions +
+
+
SORTING OF IONS +
 Quadrapole
at high vacuum +
++-
+
+
 Time of Flight
+
-  Paul Trap
SAMPLE DESOLVATION
AND IONIZATION
 EI
• Therefore the interface must:  Electrospray
LC/MS  APCI
• Remove the solvent INTERFACE
• Leave the analyte HPLC DATA PROCESSING
MASS SPECTRUM
• Charge the analyte

Dr. Shulamit Levin, Medtechnica

1
 Transition from LC to MS
Defining Data Description of Data Format
(Spectrum Format)
z From Raw Data to Mass Spectrum

• State of Matter: Liquid to Gas Instrument Basic Mass Range

Threshold Parameters Data Format
Raw Data Mass Spectrum

Neutral” to Ion
• Charge State: “Neutral Instrument Advanced Scan Time
Threshold Parameters Inter-Scan Delay

• Pressure: 760 torr to 10-5 to 10-8 torr 102,1

Scan N

Intensity

Intensity

Intensity
Scan N Σ N Scans
Centroid 103,1 Continuum MCA

100 101 102 103 100 101 102 103 100 101 102 103
m/z m/z m/z

Defining Data Description of Data Format

Centroid-
Centroid- and Continous -Spectra
All Mass Spectrometers Must:
HFN1 1 (2.958) Cn (Top,5, Ar); ME [Ev-43833,It17] (Gs,0.830,980:1400,0.50,L33,R33)
100
15127

Centroid
Scan ES+
1.77e8

• Generate ions
%

• Separate ions
0
HFN1 1 (2.958) ME [Ev-43833,It17] (Gs,0.830,980:1400,0.50,L33,R33)
100
15127
Scan ES+
6.33e7
• Detect ions
Continous

Converted
%
• Compute ion intensities
0
15085 15090 15095 15100 15105 15110 15115 15120 15125 15130 15135 15140 15145 15150 15155 15160 15165
mass

• Interpret Data

Dr. Shulamit Levin, Medtechnica

2
 Diode Array 3D Run
Mass Spectrometer 3D Run

Mix

Diode Array Chromatogram

210.00 (10.696) 1: Scan ES+

Total-Ion-Current Chromatogram
Int. 100
262.87 4.34e5

with poor resolution 246.00 with poor resolution

59.99

213.90

% 222.87

235.87

Mixture
1: Diode Array Mixture 263.87

1: Mass Chromatogram
4.65 5.05
195.98 240.88 264.85
294.00
Abs 4.65 5.05
68.92
98.85
120.80
267.91 287.01
Int.
309.02
8.62
128.82
76.87 170.92
0 nm 333.84
200 210 220 230 240 250 260 270 280 290 300 310 8.62 0
60 80 100 120 140 160 180 200 220 240 260 280 300 320 340
m/z

5.65
5.65
8.02
8.02

10.62
10.62
3.82
3.82
0.74
0.74

0 Time
2.00 4.00 6.00 8.00 10.00 0 Time
2.00 4.00 6.00 8.00 10.00

Selectivity of Mass Spectrometer Detector

Ionization Modes
Mixture
4.655.05
Extracted Ion Chromatogram
Int.
5.65
8.62
of a single Component from
8.02
a mixture of Components
0.74
3.82
10.62
I. Electron Impact-Ionization (EI)/Chemical Ionization
0 2.00 4.00 6.00 8.00 10.00 Time (CI)
Mix

II. Atmospheric Pressure Ionization (API)

10.60 1: Scan ES+
100 262.87
4.59e5

• Atmospheric Pressure Chemical Ionization (APCI)

%

• Electrospray (ESI)
0 Time
2.00 4.00 6.00 8.00 10.00 12.00 14.00

Dr. Shulamit Levin, Medtechnica

3
 MS Configuration for Ionization Electron Ionization (EI)
Filament
-
Repeller
Sample
API +
Interface
Introduction Ion to Mass Detector
Source Vacuum Analyzer Sample Inlet Ions M + e- M+. + 2e -
M
- To
High Vacuum Analyzer
F 1 F2 F3

Sample
EI Collector Ion Focusing
Lenses
Introduction Interface Ion Mass Detector
to Vacuum Source Analyzer
A beam of solute particles (Particle Beam) enter the MS source and
High Vacuum hits a heated wall where they sublime. Once in vapor phase, the molecules
are ionized by Electron Impact (EI) or Chemical Ionization (CI).

EI Spectrum
Ionization Modes

I. Electron Impact-Ionization (EI)/Chemical Ionization

(CI)

II. Atmospheric Pressure Ionization (API)

• Atmospheric Pressure Chemical Ionization (APCI)

• Electrospray (ESI)

Dr. Shulamit Levin, Medtechnica

4
 APCI APCI Mechanism

Ion formation (Aerosol/Evaporation/Ionization/Evacuation)

Ionization produces
solventions
• Heat rapidly evaporates both solvent and solutes. x
x xH + M xH + XH + M H+
• APCI creates gas-phase ions at atmospheric pressure with the Corona M x xH +
M x x
discharge. x x x x M H+
x x
• Small Gas-phase ions interact with neutrals.
C orona
• Volatile salt buffer are recommended. H eated N ebulizer N eedle The solventions react
w ith analyte m olecules
X = SolventM olecules e.g.H 2O ,M eC N form ing clusters
M = Sam ple M olecule

APCI
APCI

Ionization Process General applicability

• After evaporations of neutral species ions are formed by the Corona

• Molecule < 2000 amu
discharge process
• Ionizable, polar and mid-polar molecules
• All species in the gas phase undergo significant collisions with surrounding
• Available on all MS types
gases
• Extremely sensitive technique for compounds
• SH+ + M ----> MH+ + S (S= solvent, M= molecule)
with high proton affinity like amines

• Positive and negative ionization modes

Dr. Shulamit Levin, Medtechnica

5
 APCI

Ionization Modes
Advantages Disadvantages
• Molecular weight information • Thermal degradation may occur
• Easy to use I. Electron Impact-Ionization (EI)/Chemical Ionization
• High chemical noise at low mass
• Rugged
• Not appropriate for compounds (CI)
• Very efficient ionization
with MW above m/z 2000
• Accommodates LC flows
II. Atmospheric Pressure Ionization (API)
• Volatile buffers required
up to 2.0 ml/min
• Atmospheric Pressure Chemical Ionization (APCI)
• Good sensitivity

• Good complement to ESI • Electrospray (ESI)

(same hardware)

Electrospray Ionization
Positive or Negative?

Basic Compounds (-NH2) (M+H)+

Acidic Compounds (-CO2H, -OH) (M-H)-

Dr. Shulamit Levin, Medtechnica

6
 Recognizing Multiply Charged Ions Mass Range
Multiply Charged Molecules

Mass spectrometers operate on the basis of mass-to-charge ratio (m/z). Horse Heart Myoglobin
Mass assignments are normally made assuming a single charge per ion n = 23, m/z = 738
(i.e. m/z = m) n = 22
n = 21
n = 20
n = 19
Single charge Mass = (M+H) n = 18
n = 17
Double charge Mass = 1/
2 (M+2H) n = 16, m/z = 1060
n charge Mass = 1/n (M+nH)

Isotopes of doubly charged ions are separated by 0.5 Da

Acquired Mass range Calculated Mass

Hemoglobin Spectrum Deconvolution by MaxEnt

Presence of More Than One Charged Envelope Hemoglobin
100 15125.0
1081.60

100
1164.52

1009.36

1261.64

%
% 1376.08 15857.0
1133.92 1221.18 15866.0
1323.14
1058.83

15149.0

0 mass
0 m/z 15000 15100 15200 15300 15400 15500 15600 15700 15800 15900 16000 16100
1000 1050 1100 1150 1200 1250 1300 1350 1400

Dr. Shulamit Levin, Medtechnica

7
 Multiply Charged Ions
(M+H)++
(M+2H)2+
Advantages
• MW confirmation
• High MW determination
• Volatile & non-volatile solutes
• “No limit” in MW
• Ionic/polar analytes
• low temperature, no degradation
• Good sensitivity
• Quantitative method
• Suitable for CapLC
Dr. Shulamit Levin, Medtechnica
ESI General applicability
1 Da
• Small singly charged ions.
• Large ionic molecules.
• Virtually any ion in solution is a candidate for ESI
(+ or - ions).
0.5 Da • The production of multiple charge ions (proteins) extends
the mass range detection to no limit.
• Available on all MS types
• Most sensitive LC/MS technique
• Molecular weight
• Structure with MS/MS or CID
Electrospray Ion Separation Analyzers
Disadvantages
• Requires low LC flow rates for best aerosol 1. The Quadruple
• Must form ions in solution
2. Time of Flight
• Ion suppression with high salt condition
• Adduct ions may result
3. Ion Trap
• Limited structural information 4. Magnetic sector
• Users must understand chemistry of aqueous
solution (acid-base equilibrium, redox chemistry..)
5. Fourier Transform Ion Cyclotron
• Unknowns should be analyzed in both positive and
negative ion modes.
8
 TT im
im ee O
O ff FF lig
lig hh tt M
M aass ss A
A nn aa ly
lyzz eerrss
FT-
FT-ICR-
ICR-Spectrometer
Magnetic Fielt B
SSO
OUURRC
CEE R
REEFF LLE
ECC TTR
ROON
NOOF
F FF
DDRRIF
IFTT TTUUBBEE
DDEETTEEC
LLIN
INEEA
CTTO
ORR
ARR M
MOODDEE Starting with the quadrupole
DDEETTEECCTTO
ORR
RREEFFLLEEC
CTTRRO
ONN M
MOODDEE

Z
X Trapping Plates SSO
OUURRC
CEE R
REEFF LLE
ECC TT R
ROON
NOON
N
Source Elektroden Electrodes DDEETTEECCTTO
ORR
DDRRIF
IFTT TTUUBBEE
DC LLIN
INEE A
ARRMMOODDEE
Sender

DDEETTEECCTTOORR
Receiver Plates
RREEFFLLEECCTTRRO
ONN M
MOODDEE

Filament

DC
DC
Resonant Ion
Ion Traps
Transferoptic Transmitter Plates

199 Nonresonant Ion

Detector
End Cap Electrode

Types of Axial Modulation

Mass Spectrometer’s +
+ + +
+ +++
Ring Electrode, Rf

Analyzers Inlet

Electron Multiplier
Ωt
V(t)= -V dc -V rf cosΩ
The Quadrupole Analysator 190

S ector M ass S pectrom eters

resonant Ion
non resonant Ion
Nie r-Jo h ns o n-G eo m etry (EB )
Ωt
V(t)= V dc + V rf cosΩ
Slit
Detector

M a gn etic s ec tor

Elec tro static Sec to r

(ES A)

dc and Rf Voltages
D etec tor dc and Rf voltages
Slit

Ion Source Io n

Source
So urce

77

220

Fragmentation of Molecules for Identification Triple Quadrupole (MS/MS)

Tandem-in-Space

• Precursor ions and product ions are created and analyzed in

different physical spaces.
• Ions must be moved from "source" to analyzer (different
physical regions) where different functions take place.

Ion Q1 Q2 Q3
Source CID
Region

Dr. Shulamit Levin, Medtechnica

9
 Typical Specifications Time of Flight Mass Analyzers

• Quantification: best choice for solving

SOURCE
REFLECTRON OFF DETECTOR
• Mass Range: 2-4000 u DRIFT TUBE
LINEAR MODE
quantification problems.
• Resolution: Unit Resolution • Variations: single QuadSIR/scanning
DETECTOR
Triple Quad-SRM/scanning Hybrids, Q- REFLECTRON MODE
• Mass Accuracy: ca ±0.1 u TOF

• Scan Speed: 4000 u/s (in practice • Positive and negative Ions SOURCE REFLECTRON ON
DETECTOR
• References: Miller, P.E., Denton, M.B., DRIFT TUBE
LINEAR MODE
500 - 1000 u/s)
J.Chem Educ. 7, 617, (1986).
DETECTOR
• Vakuum: 10-4 up to 10-5 Torr REFLECTRON MODE

Typical Specifications TOF

Time of Flight

• A package of ions is accelerated by a potential V into a field free flight tube. • Mass Range: 2-15000, high • Quantification: low dynamic range
•The time t fight needed for ions to reach a detector placed at distance d is • Variations: Linear and Reflectron, Tandem
sensitivity
measure: with quads and sectors
m/z = [k V /d 2] t 2 • Resolution: 10000 (Reflectron) • Positive and negative Ions : ESI, APCI,
fight
•Small ions have higher velocity than large mass ions therefore their time of flight • Mass Accuracy: ≈5 ppm MALDI
in the tube are different and related to m/z ratio. • References: Cotter, R.J., “Time-of-Flight:
•A high percentage of the generated ions is detected therefore • Scan Speed: very high 106 u/s,
Instrumentation and Applications in
this technology provides very high sensitivity. Biological Research, ACS: Washington, D.C.
sampling rates 300-500 MHz.
•Fast scanning capabilities (>106 dm/dt) and no limit in mass range. 1994.
• Vakuum: 10-7 Torr
•However it requires very high vacuum (10-7 torr).

Dr. Shulamit Levin, Medtechnica

10
 5ppm - So What?
Where Exact Mass becomes really
interesting...
• Differentiation of nominally isobaric metabolites
• 2 organic molecules may have the same nominal mass but different • For example…
– Dehydroxylation followed by methylation...
elemental composition
• R-CH2-OH → R-CH3(-O)
• An exact mass measurement may be able to show this • R-OH → R-OCH3(+CH2) (-O+CH2 = -1.9793 Da)
• This becomes useful in
– metabolism studies – Alcohol to Aldehyde ….
(-H2 = -2.0157 Da)
– impurity studies • R-CH2-OH → R-CH=O

– environmental analysis • CO = 27.9949

– synthetic chemistry
– industrial applications
– …………………...
• N2 = 28.0061 • Difference in m/z between the 2 metabolites is 0.0364 Da
• C2H4 = 28.0313 • At m/z 500 this is a 73ppm difference
• 5ppm will allow you to correctly ID the metabolite
• These have the same nominal mass but
different exact mass

Mass Measurement Accuracy Power and Limitations of Exact Mass

• For C0-100 H3-74 O0-4 N0-4

Component Calculated Measured +/- mDa +/- ppm
m/z m/z
Parent 360.1382 360.1366 1.6 4.4 • Mass 118 : closest neighbour at 34ppm
Sulphide 344.1433 344.1424 0.9 2.6 • Mass 500 : 5 compounds within 5ppm
Sulphone 376.1331 376.1330 0.1 0.3
• Mass 750.4 : 626 compounds within 5ppm

Desmethyl 346.1225 346.1218 0.7 2.0

• Note 5ppm at m/z 500 means m/z 500 ± 0.0025
S-Desmethyl 330.1276 330.1265 1.1 3.3
Aldehyde 344.1069 344.1074 0.5 1.5
(reference J. Am. Soc. Mass Spectrom.)
S-Pyridone 272.0858 272.0867 0.9 3.3

Dr. Shulamit Levin, Medtechnica

11
 Ion Trap
Q-TOF Hybrid Mass Spectrometer

PUSHER DETECTOR

ESI PROBE
QUADRUPOLE
SAMPLING CONE
SKIMMER HEXAPOLE
COLLISION CELL

RF HEXAPOL

REFLECTRON

QTof:
QTof: ”Quadrupole-TOF Tandem"

Ion Trap MS/MSn Typical Specifications IT

• Mass Range: 20-2000 u • Quantification: possible, but not very

Accumulation Isolation
accurate
• Resolution: Unit Resolution • Variations: MS/MS, Sources- Internal and
External, Resonance Excitations
• Mass Accuracy: ca ±0.1 u • Positive and negative Ions
Fragment Excitation • References: March, R.E., Huges, R.J.
Accumulation • Scan Speed: 4000 u/s
“Quadrupole Storage MS”, John Wiley and
Sons: New York, 1989.
Vakuum: 10-3 Torr

Detection Fragmentation

Dr. Shulamit Levin, Medtechnica

12
 Sector Mass Spectrometers Typical Specifications - Sector

Nier-Johnson-Geometry (EB) Slit

Magnetic sector • Mass Range: 20000u Resolution: • Variations: 1-, 2,-, 3-, 4-Sectors
Electrostatic Sector Tandem with quads and TOF
with double focusing: 100000
(ESA)
• Positive and negative Ions : ESI, APCI, EI,
• Mass Accuracy: ≈1 ppm CI, PB, Continuos flow FAB
Detector

• Scan Speed: slow • References: Dass, Chhabil “Chapter 1

Slit
Magnet
Instrumentation and Techniques,” IN: Mass
Ion
• Vakuum: 10-7 Torr

ease
Incr

m/z
Source 4 Spectrometry: Clinical and Biomedical

d
3
Ion beam 2
1
The radius con be obtained from:
Applications; Desidero, D.M. (Ed.), Plenum,
r = √ m (2 V/z B2)
New York (1994).
V = accelerating potential applied to ions leaving the source
B = magnetic field strength

FT-
FT-ICR-
ICR-Spectrometer Typical Specifications – FT-
FT-ICR

Magnetic Field B

Y • Mass Range: > 15000 • Quantification: possible, but not very

accurate
Z
X Trapping Plates • Resolution: High, 106 • Variations: MS/MS, Sources- Internal and
Source Elektroden Electrodes
Sender
DC External, SWIFT: Stored Waveform Inverse
• Mass Accuracy: up to 100 ppb
FT
Receiver Plates
• Scan Speed: fast ms Vakuum: 10• - Positive and negative Ions
Filament
• References: Buchanan, M.V. (Ed.), “Fourier
DC
7 - 10-9 Torr
DC
Transferoptic
Transform MS”: American Chemical
Transmitter Plates
Society: Washington D.C, 1987.

Dr. Shulamit Levin, Medtechnica

13
 Photomultiplier
Electron Multiplier
high voltage conversion dynode (converts either positive or negative
A conversion dynode is used to convert either negative or ions into electrons)
positive ions into electrons. Higher potentials on the conversion electrons impinge onto light emmitting phosphor that is optically
dynodes are used to accelerate high mass ions and thereby coupled to a photomultiplier

enhance sensitivity. photomultiplier permanently sealed in its own glass envelope. The
detector is protected from contamination and thus longer lifetimes are
achieved.
- Conversion Dynode
+ + - (Voltage 1- 20 kV) The photomultiplier has a 10 year maintenance-free lifetime.
Mass Analyser + + + +
-
Electron Multiplier dynode

(voltage setting lower than phosphor

photom ultiplier
Dynode)

Current is measured

Mass Electrospray Produces Multiple Charging

(consequently high MWs can be measured)

2+
M3+ M
Nominal Mass
Ion Series
The mass of an ion with a given empirical formula calculated using the integer Ion m/z M4+ M+
mass numbers of the most abundant isotope of each element
Ex : M=249 C20H9+ or C19H7N+ or C13H19N3O2+ M+ X/1 M5+
M2+ X/2
M3+ X/3
Exact Mass M/4+ X/4
The mass of an ion with a given empirical formula calculated using the exact M5+ X/5
mass of the most abundant isotope of each element
Ex : M=249 C20H9+ 249.070 X/5 X/4 X/3 X/2 X
C19H7N+ 249.0580
C13H19N3O2+ 249.1479 Mass/Charge
Multiply charged ion series is the key to determining molecular mass with Electrospray MS

Dr. Shulamit Levin, Medtechnica

14
 Resolution

Mass Range
Multiply Charged Molecules Resolution, equally called Resolving Power, of a mass
spectrometer is a measure of its ability to separate
adjacent ions.
5 uM Cytochrome c (horse heart)
8,000
Positive ion mode,
[M+16H]16+ MW = 12,360.9 ESI, 2 ul/min,
At higher resolution, small may differences may be detected.
50% MeOH:49%
6,000 [M+17H] 17+ [M+15H] 15+
C20H9+
H2O:1% HOAc
C19H7N+
Intensity

C13H19N3O2+ C20H9+ C19H7N+ C13H19N3O2+

[M+14H] 14+
4,000

2,000 18+ [M+13H]

13+ 3 different compounds 3 different compounds
[M+18H]
Same nominal mass 3 different exact masses
Low resolution High resolution
0
650 700 750 800 850 900 950 1,000
m/z
249 249.0700 249.0580 249.1479

Determining Resolution
Defining Mass Resolution
FWHM Resolution Example

P P G 2 0 0 0 R es o lu tio n
mave p pg (0.5 1 7) C u ( 0.2 5 ); Is (1 .0 0,1 .00 ) C 10 2 H 2 10 O 3 5N
20 10.47
Sca n ES +
3 .40 e 1 2
1 00
∆m=0.25 amu
2009 .47
∆mr
Single Ion method Double Ion method 50% 2011.47 FWHM~8040
%

Full Width at Half Maximum 2 adjacent ion peaks 201 2.4 7

(FWHM) with a 10% valley max 2013 .47

or at 5% of the peak height 0
201 4.4 7

mave p pg (0.5 1 7) C u ( 0.5 0 ); Is (1 .0 0,1 .00 ) C 10 2 H 2 10 O 3 5N

1 00
20 10.47
∆m=0.5 amu
Sca n ES +
3 .42 e 1 2

m R= 2009 .47

R= ∆mr
∆m 50% 2011.47
FWHM~4020
%

201 2.4 7

2013 .47

0 m a ss
2 00 7 20 0 8 2 0 09 2 01 0 2 0 11 20 1 2 2 01 3 20 1 4 2 0 15 2 01 6

Dr. Shulamit Levin, Medtechnica

15
 Mass Accuracy Mass Accuracy vs Resolution

Ability of a mass analyzer to assign the mass

of an ion close to its true value (exact mass)

∆m accuracy = mreal - mmeasured

∆maccuracy
ppm = 106 * ∆m accuracy / mmeasured

High mass accuracy (exact mass measurement)

is usually associated to high resolution analyzers
Goals :
- Unknown compound determination
Exact mass helps to define its atomic composition

- Target analysis
Exact mass proves the presence of a particular
ion in a mixture

Sensitivity Scan Speed (or rate)

• Term used to describe the ability of the MS to respond to a given amount of

sample analyte at a given mass to charge ratio (compound dependent) • The rate at which we can acquire a mass spectrum, (mass units/sec).
• Is an essential acquisition parameter for MS
• Mass Spectrometers are mass-flow sensitive device.
Sensitivity = Area( or height) • Will affect the amount of information (qualitative and quantitative) that
Cmax x flow rate can reasonably be attained with a given mass analyzer.

• The sensitivity of your LC/MS system is directly related • Minimum Scan Rate:
to the efficiencies of all the processes of the entire system :
- LC separation and flow dm/dt 10 points = 10∆macquisition / WLC peak
- Interface
- Ionization Cmax = 4 M (N)1/2
- Mass transmission π d2 ε L (1+k’) (2π
π)1/2
- Detection

Dr. Shulamit Levin, Medtechnica

16
 Triple Quadrupole

Q1 Q2 Q3
A Typical Bio-
Bio-Analytical Quantitative Study Using Triple
Full Scan
MS
Quadrupoles:
Quadrupoles:

Measurement of Amphetamines in Human Saliva

SIM
by LC-
LC-MS/MS
MS/MS
Product
Ion Mode

MS/MS
SRM michelle.wood@micromass.co.uk

MS Scan of Ecstasy (MDMA) Specificity = MRM

Parent 194 Daughter 163

CH 3
HN
CH 3 O O
HN

H 3C O H 3C O

Product ion Scan of m/z 194

M/Z Collision energy = 12eV, Argon = 2.5x10-3 mbar

Dr. Shulamit Levin, Medtechnica

17
 Signal:Noise for Carbosulfan Calibration Graph for Carbosulfan

Compound name: Carbosulfan

(0.25pg/µL std) Coefficient of Determination: 0.999380
Calibration curve: 273692 * x + 6294.89
Response type: External Std, Area
Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

6926597

Response

0 pg/µl
0.0 5.0 10.0 15.0 20.0 25.0

Nominally isobaric metabolites -

Q-TOF Hybrid Mass Spectrometer-
Spectrometer- rabeprazole
Peptide & Protein Sequencing O

O
PUSHER DETECTOR
O N
S H
N
N
ESI PROBE O H O
QUADRUPOLE
SAMPLING CONE O
SKIMMER HEXAPOLE O
COLLISION CELL

N O N
RF HEXAPOL
S S
N N
N N
REFLECTRON H H

QTof:
QTof: ”Quadrupole-TOF Tandem" Difference = 36 mDa
Sulphide Aldehyde
[M+H]+ 344.1433 [M+H]+ 344.1069

Dr. Shulamit Levin, Medtechnica

18
 Will MS/MS help? Exact mass chromatograms
O

O m/z 226.0902
H+ ( 4.1 ppm)
N N
O
S
N N
N H O
H
m/z 119.0609
( 7.5 ppm) S+ 1 Da window
Sulphide N
m/z 344.1433

H
Aldehyde
O
m/z 226.0538 0.03 Da window
+ H
O H ( 2.7 ppm)
N
O
O N N
S H O
N Sulphide
N
H m/z 119.0609 O
( 5.9 ppm) S+
0.03 Da window
N
Aldehyde
m/z 344.1069

Sulphide + Aldehyde Metabolites

Exact Mass is important to Multiply Charged Ions
O
O

N
S
N N
H
(M+H)++
Monoisotopic M/Z
Sulphide +/- 1 Da

m/z 344.1433 ( 2.6 ppm)

H
O
O
O N
S (M+2H)2+
2+
N Monoisotopic M/Z
N
H +/- 0.5 Da

Aldehyde
m/z 344.1069 ( 2.0 ppm)

Dr. Shulamit Levin, Medtechnica

19
 Peptide and Protein Sequencing Strategy Followed in Protein Identification using Mass
Spectrometry

FRACTIONATION
y3 y2 y1 OF PROTEIN
CHEMICAL OR
ENZYMATIC
MIXTURE DIGESTION
+2H +2H +2H e.g. tryptic
e.g. by gel
R1 R2 R3 R4 electrophoresis digest

H2 N CH CO NH CH CO NH CH CO NH CH COOH
COMPLEX UNKNOWN PEPTIDE ESI-MS
IDENTIFICATION OF
a3 PROTEIN INTACT ANALYSIS
a2 MIXTURE e.g. nanoflow PROTEIN
PROTEIN

b1 b2 b3
Nomenclature of Peptide PEPTIDE
Computer search of
the experimentally
SAMPLE determined
Fragment Ions CLEAN-UP
e.g. by LC
molecular weights
against a database of
peptide fragments

Drug Discovery Process

Identification of Target
Nanoscale LC-
LC-ESI-
ESI-MS-
MS-MS
ADH 250fmoles inj.
HV001 78 (22.825) Cm (78:81) 2: TOF MSMS 536.34ES+
487 112
100 586 815
240
228 355
260 456

% 341
440
816
MS-MS spectrum
373 587 699
222 385
327 527 555 701
428
695 816
129 147 201 412 667 713 794
510 926
824 943
0 m/z
100 200 300 400 500 600 700 800 900 1000 1100 1200

ADH 250fmoles inj.

HV001 1: TOF MS ES+
100 28.38 BPI
779

13.81

27.11
18.92
24.66

%
Chromatogram
19.32
21.63

22.17

17.41
17.50 20.10

21.09
22.87
15.24
26.30

16.51 31.73
31.02

0 Time
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00

Dr. Shulamit Levin, Medtechnica

20
 Unit mass MS/MS
Data Directed Analysis (DDA)

DG020118_03 7 (0.616) TOF MSMS 1465.80LD+

1465.84
100 355.19
31

159.10 483.25
200.11

325.20
%
386.20
315.16 581.36 598.39
298.13 554.30
424.26
211.15
535.29 868.43
158.10 1448.82
652.30 691.40 740.38 912.52 1080.60 1266.72

0
DG020118_02 94 (8.118) TOF MSMS 1464.70LD+
100 1464.79 26
323.16

667.31 780.41
649.32
% 509.24 572.30
305.15

685.38 1465.84
355.19 596.28
175.13 371.22 481.25
159.10 225.11 458.25
296.17 763.38 798.48 869.51 956.58 1446.79
110.08 424.25
1080.64 1142.62 1279.65
1057.85 1265.25

0 m/z
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400

1464.8 - MSMS
Searching over the Internet

Dr. Shulamit Levin, Medtechnica

21