Mass Spectrometry - Instruments

Genomic and Proteomic Research

• Genomic research: – Research on genomes.

– Defines potential contributors to cellular functions

• Proteomic Research: – Research on the comprehensive group of proteins 

expressed by a given cell or tissue. 

– Expressed genome defines actual contributors.

Central tools in proteome research

• SDS‐

PAGE– 1D and 2D gel electrophoresis

• Protein sequencing– Edman degradation 

– Mass Spectrometric technique (Tandemm and  Maldi

• Identification techniques– Immunologic technique (Western blotting)

Western blotting

• Identifies a protein based on pattern of  antibody recognition. 

• Presumptive and require the availability of a  suitable antibody 

• The confidence of the identification is limited  by problems with the specificity of the 

antibody

Tandem Mass‐Spectrometry

• Cut bands of interest directly from the gel,  digest, analyze and identify

• Advantages: – High sensitivity: femtomole level

– Rapid speed of analysis– Large amounts of information generated in each 

experiment

– Ability to characterize post‐transitional  modifications

Mass Spectrometer Schematic

Inlet Ion Source

MassFilter Detector Data

System

High Vacuum System Turbo pumpsDiffusion pumpsRough pumpsRotary pumps

Sample PlateTargetHPLCGCSolid probe

MALDIESIIonSprayFABEI/CI

TOFQuadrupoleIon TrapMag. SectorFTMS

Microch plateElectron Mult.Hybrid Detec.

PC’sUNIXMac

HPLC/MSD Unit

11/15/2010 Iowa State University - Dong Ahn 7

LC/ESI‐Ion Trap MSD 

11/15/2010 Iowa State University - Dong Ahn 8

A simple definition of a Mass Spectrometer 

• A Mass Spectrometer is an analytical instrument that  can separate charged molecules according to their 

mass–to–charge ratio. 

• The mass spectrometer can answer the questions 

– “what is in the sample”

(qualitative

structural information) 

– “how much is present”

(quantitative

determination) for a  very wide range of samples at high sensitivity 

10

MS Principles

• Different elements can be uniquely identified  by their mass

MS Principles

• Different compounds can be uniquely  identified by their mass

CH3 CH2 OH

NOH

HO

-CH2 -

-CH2 CH-NH2

COOHHO

HO

Butorphanol L-dopa Ethanol

MW = 327.1 MW = 197.2 MW = 46.1

How are mass spectra produced ? 

• Ions are produced in the source and are  transferred into the mass analyser 

• They are separated according to their  mass/charge ratio in the mass analyser (e.g. 

Quadrupole, Ion Trap) 

• Ions of the various m/z values exit the  analyser and are ‘counted’

by the detector 

What is a Mass Spectrum?

• A mass spectrum is the relative abundance of  ions of different m/z produced in an ion 

source – A “chemical fingerprint”

• It contains – Molecular weight information (generally) – Structural Information (mostly) – Quantitative information 

What does a mass spectrum look like ? 

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

Mass Spectrometry

• For small organic molecules the MW can be  determined to within 5 ppm or 0.0005% which is 

sufficiently accurate to confirm the molecular  formula from mass alone

• For large biomolecules the MW can be determined  within an accuracy of 0.01% (i.e. within 5 Da for a 

50 kD protein)

• Recall 1 dalton = 1 atomic mass unit (1 amu)

MS History

• 1948‐52 ‐

Time of Flight (TOF) mass analyzers  introduced

• 1955 ‐

Quadrupole ion filters introduced by W. Paul,  also invents the ion trap in 1983 (wins 1989 Nobel 

Prize)

• 1968 ‐

Tandem mass spectrometer appears

• Mass spectrometers are now one of the MOST  POWERFUL ANALYTIC TOOLS IN CHEMISTRY

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?

• 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.

How is mass defined?

Lecture 2.1 20

Resolution & Resolving Power

• Width of peak indicates the resolution of the MS  instrument

• The better the resolution or resolving power, the  better the instrument and the better the mass 

accuracy

• Resolving power is defined as: M/ΔM– M is the mass number of the observed

– ΔM is the difference between two masses that can be  separated 

Monoisotopic mass

Monoisotopic masscorresponds tolowest mass peak

When the isotopes are clearly resolved the monoisotopic mass

is used as it is the most accurate measurement.

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.

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

100

0

10

20

30

40

50

60

70

80

90

100

% In

tens

ity

ISO:CH3

15.0229

M

FWHM = ΔM

R = M/ΔM

How is mass resolution calculated?

Advantage of using high resolution  mass spectrometry

25

MS Principles

• Find a way to “charge”

an atom or molecule (ionization)– Generate gas‐phase ions derived from an analyte (protein)

– Measure the mass‐to‐charge (m/z) of those ions

• Place charged molecule in a electromagnetic field and  measure its speed or radius of curvature relative to its  mass‐to‐charge ratio (mass analyzer)

• Gas‐phase ion– can be precisely controlled in electromagnetic field that contain the 

ions for study

– Can be manipulated to probe the movement of the ions in the field

• Detect ions using microchannel plate or photomultiplier  tube

Mass Spec Principles

Ionizer

Sample

+_

Mass Analyzer Detector

Different Ionization Methods

• Electron Impact (EI

‐

Hard method)

– small molecules, 1‐1000 Daltons, structure

• Fast Atom Bombardment (FAB

– Semi‐hard)

– peptides, sugars, up to 6000 Daltons• Electrospray Ionization (ESI

‐

Soft), Atmospheric Pressure 

Chemical and photoionization (APPI and APCI ‐

Soft)

– peptides, proteins, up to 200,000 Daltons• Matrix Assisted Laser Desorption (MALDI‐Soft)

– peptides, proteins, DNA, up to 500 kD

Comparison of Ionisation Techniques

Electron Impact Ionisation

• Widely used technique when coupled to GC

• Suitable for volatile organic compounds– e.g. hydrocarbons, oils, flavours, fragrances

• Energetic process – A heated filament emits electrons which are accelerated by a 

potential difference of usually 70eV into the sample chamber

• Molecule is “shattered”

into fragments (70 eV >> 5 eV bonds)

• Ionisation of the sample occurs by removal of an electron  from the molecule

• Produces M+. 

radical cation giving molecular weight

• Produces abundant fragment ions

• Fragments sent to mass analyzer

• Library searchable spectra

EI Fragmentation of CH3

OH

CH3 OH CH3 OH+

CH3 OH + CH2 O=H+ + H

CH3 OH +CH3 + OH-

CHO=H+ + H+CH2 O=H+

Electron Ionisation

Chemical Ionisation

• Similar ionisation technique to EI except that a reagent  gas is introduced into the chamber in excess of the 

sample• Positive CI uses methane, isobutane or ammonia as 

reagent gases• Negative CI uses methane as a reagent gas in electron 

capture mode• Ionised reagent gas protonate the sample molecules 

leaving a neutral reagent gas species• Softer ionisation technique• Positive mode generates ions of M + H

• Negative mode generates ions of M ‐

H• Not reproducible from lab to lab.

Chemical Ionisation

Chemical Ionisation

Why You Can’t Use EI For Analyzing  Proteins

• EI shatters chemical bonds

• Any given protein contains 20 different amino acids

• EI would shatter the protein not only into amino acids but  also amino acid sub‐fragments and even peptides of 

2,3,4…

amino acids

• Result is 10,000’s of different signals from a single protein ‐ ‐

too complex to analyze

Soft Ionization 

• Soft ionization techniques keep the molecule of interest  fully intact

• Electro‐spray ionization was first conceived in 1960’s by  Dole but put into practice in 1981 by Barber (FAB) in 1985 

by Fenn (ESI)

• MALDI first introduced in 1988 by Hillenkamp and Karas 

• Made it possible to analyze large molecules via  inexpensive mass analyzers such as quadrupole, ion trap 

and TOF

Lecture 2.1 37

Fast Atom Bombardment

• Used for large compounds with low volatility (e.g.,  peptides, proteins, carbohydrates)

• Solid or liquid sample is mixed with a non‐volatile  matrix (e.g., glycerol, crown ethers, nitrobenzyl 

alcohol) 

• Immobilised matrix is bombarded with a fast beam of  Argon or Xenon atoms. 

• Charged sample ions are ejected from the matrix and  extracted into the mass analysers

• Gives (M+H)+, (M‐H)‐

or (M+Na)+ ions

• Choosing correct matrix is difficult

Fast Atom Bombardment Source

Atmospheric Pressure Ionisation 

• Most important and widely used LC / MS   technique 

• API two types – Electrospray  – Atmospheric Pressure Chemical Ionisation 

• > 99% new LC/MS use API source • Ionisation takes place outside vacuum region

Atmospheric Pressure Ionisation 

• API coupled to LC or CE or Nanospray • Handle wide range of flow rates • Produce Intense (M+H)+ ions • Very little fragmentation 

‐

Need MS/MS for structural information 

• Applicable to wide range of compounds • Sample must be in solution 

Advantages of API

• Soft ionisation (gives the molecular weight)

• Sensitive (low pg amounts routinely)

• Robust, simple, run routinely 24 hr/day

• Wide range of flow rates (nanospray to analytical)

• Wide range of applications (drugs, proteins)

• Wide range of industries

Electrospray Ionization (ESI)

• Softest ionisation technique

• ESI is a liquid phase ionization process

• Best for polar non‐volatile compounds (proteins, peptides,  nucleic acids, Pharmaceuticals, natural products)

• Can produce multiply charged ions allowing  determination of high molecular weight proteins

• Ions are ejected from charged vapour droplets to gas  phase producing (M+H)+ or (M‐

H)‐

ions

• Strongly affected by salts & detergents

• Not very tolerant of non‐volatile salts

Electrospray Ionization

• Sample dissolved in polar, volatile buffer (no salts) and  pumped through a stainless steel capillary (70 ‐

150 μm) 

at a rate of 10‐100 μL/min• Coupled to LC at a flow range of 2‐1000 ul/min, 

nanospray (10 nL/min – 2 uL/min)

• Strong voltage (3‐4 kV) applied at tip along with flow of  nebulizing gas causes the sample to “nebulize”

or 

aerosolize

• Ion formation occurs as the droplets in the aerosol  decrease in size resulting in ions being ejected into the 

gaseous phase

• ESI is a concentration dependent process

Electrospray ion source

• The LC eluent is sprayed (nebulized) into a chamber at atmospheric pressure in the presence of a strong electrostatic field and heated drying gas.

• The heated drying gas causes the solvent in the droplets to evaporate.

Desorption of ions from solution

++++ +

++++

++ + +

++++

++

+++

Liquid5 kv

+++++

+++

++

+++++

++++

+

++ + + + ++

+++++++

+

++ ++

+++ +

Droplet

Formation

Droplet

Evaporatio

n

Coulombic

Explosion

Ion Evaporation

Molecularion

H+

+++++

• As the droplets shrink, the charge concentration in the droplets increases.• The repulsive force between ions with like charges exceeds the cohesive

forces and ions are ejected (desorbed) into the gas phase. • Useful for analyzing large biomolecules

Electrospray Ionization

• Can be modified to “nanospray”

system with flow < 1 mL/min

• Very sensitive technique, requires less than a picomole of material

• Strongly affected by salts & detergents• Positive ion mode measures (M + H)+

(add formic

acid to solvent)• Negative ion mode measures (M -

H)-

(add ammonia

to solvent)

Positive or Negative Ion Mode?

• If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive

ion

detection is used

• Acidic conditions protonate all basic side chains (Lys, Arg, His) and N-terminus -

(Acetic acid

increase ionization while trifluoroacetic acid suppresses it)

• If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative

ion detection is used

Peptide Masses From ESI: Multiply Charged Ion

m/z = (MW + nH+)n

m/z = mass-to-charge ratio of each peak on spectrumMW = MW of parent moleculen = number of charges (integer)H+ = mass of hydrogen ion (1.008 Da)

Each peak is given by:

ESI spectrum ofHEW LysozymeMW = 14,305.14

Peptide Mass Calculation From ESI

1431.6 = (MW + nH+)

n

Charge (n) is unknown, Key is to determine MWChoose any two peaks separated by 1 charge

1301.4 = (MW + [n+1]H+)

[n+1]

2 equations with 2 unknowns - solve for n first

n = 1301.4/130.2 = 10

Substitute 10 into first equation - solve for MW

MW = 14316 - (10x1.008) = 14305.9 14,305.14

Atmospheric Pressure Chemical Ionization  (APCI)

• Used for wide range polarity of compounds

• HPLC eluent (up to 2ml/min flow rate) is vaporized at up to 600 C

• The Corona discharge needle ionizes solvent molecules

• A combination of collisions and charge transfer reactions between the solvent and the analyte results in the transfer of a proton to form either (M+H)+ or (M-H)- ions

• Compounds can be thermally degraded

• Multiply charged ions are rare

• More tolerant to salts

APCI Ion Source• The LC eluent is sprayed

through a heated (250°C – 400°C) vaporizer at atmospheric pressure.

• The gas-phase solvent molecules are ionized by electrons discharged from a corona needle.

• The solvent ions then transfer charge to the analyte molecules through chemical reactions (chemical ionization).

• The analyte ions pass through a capillary sampling orifice into the mass analyzer.

APCI ‐

Schematic

. . . . . ..

. . . . . ..M

S

S

SH

MSH

S

MH

SH

S

Ionization producessolvent ions

Corona DischargeNeedle

Solvent ions react with analyte moleculesto form analyte ions by charge transfer

MHMHSH

SHS

APCI +ve ion mode

APPI Ion Source

• Vaporizer converts the LC eluent to the gas phase.

• A discharge lamp generates photons.

• The energy ionizes analyte molecules while minimizing the ionization of solvent molecules.

• The resulting ions pass through a capillary sampling orifice into the mass analyzer.

Matrix Assisted Laser Desorption Ionisation 

• Similar process to FAB 

• Sample is dissolved in matrix, which absorbs light  from a short pulse of laser of a specific wavelength.  

• The sample becomes ionised and extracted towards  the mass analysers 

• Coupled to Time of Flight MS 

• Not coupled to LC • High mass range achievable 

• Calibrants may be external or included in sample 

• Reproducibility issues 

MALDI Ionization

++

+

+

-

--

++

+

+

-

---++

Analyte

Matrix

Laser

+++

• Absorption of UV radiation by  chromophoric matrix and 

ionization of matrix

• Dissociation of matrix, phase  change to super‐compressed gas, 

charge transfer to analyte  molecule

• Expansion of matrix at supersonic  velocity, analyte trapped in 

expanding matrix plume  (explosion/”popping”)

+

+

+

MALDI

• Unlike ESI, MALDI generates spectra that have just a singly  charged ion

• Positive mode generates ions of M + H

• Negative mode generates ions of M ‐

H

• Generally more robust than ESI (tolerates salts and  nonvolatile components)

• Easier to use and maintain, capable of higher throughput

• Requires 10 μL of 1 pmol/μL sample

MALDI Sample Limits

• Phosphate buffer < 50 mM

• Ammonium bicarbonate < 30 mM

• Tris buffer < 100 mM

• Guanidine (chloride, sulfate) < 1 M

• Triton < 0.1%

• SDS < 0.01%

• Alkali metal salts < 1 M

• Glycerol < 1%

Separate ions based on their mass-to-charge ratio (m/z)

Operate under high vacuum (keeps ions from bumping into gas molecules)

Key specifications are resolution, mass measurement accuracy, and sensitivity.

Several kinds existQuadrupole

Time-of-flight

Ion traps are most used.

Mass analyzers

Quadrupole Mass Analyzer

• Consists of four parallel metal rods with different charges

• Uses a combination of radio frequency and DC

• voltages to operate as a mass filter.

• Lets one mass pass through at a time.

• All other ions are thrown out of their original path

• Can scan through all masses or sit at one fixed mass

Hyperbolic Quadrupoles

mass scanning mode

m1m3m4 m2

m3

m1

m4

m2

single mass transmission mode

m2 m2 m2 m2m3

m1

m4

m2

Quadrupoles have variable ion transmission modes

Principle of Time of Flight Analyzer

• A uniform electromagnetic force is applied to all ions at the same time, causing them to accelerate down a flight tube.

• Lighter ions travel faster and arrive at the detector first, so the mass-to- charge ratios of the ions are determined by their arrival times.

• Time-of-flight mass analyzers have a wide mass range and can be very accurate in their mass measurements.

Mass Spec Equation (TOF)

mz

2Vt2=

m = mass of ion L = Flight tube lengthz = charge of ion t = time of travelV = voltage

L2

Flight time and transients/second as a function  of mass*

TOF mass calibration

Ion Trap 

• Consists of a circular ring  electrode plus two end caps 

that together form a  chamber. 

• Ions entering the chamber  are “trapped”

there by 

electromagnetic fields.• Scanning field can eject ions 

of specific m/z • Advantages 

– MS/MS/MS….. – High sensitivity full scan 

MS/MS 

Ion Trap Mass Analyzer

Top View

Cut away side view

Ion Trap Mass Aanalyzer

Comparison of Quads and Traps 

SRM: selected reaction monitoringSingle Ion Monitoring (SIM)

Tandem Mass Spectrometry

72

InletInlet DetectDetectMass

AnalyzeMass

AnalyzeIonizeIonize

MSMS

InletInlet FragmentFragmentMass Analyze

Mass AnalyzeIonizeIonize

MassAnalyze

MassAnalyze DetectDetect

MS1MS1 CollisionCell

CollisionCell MS2MS2

MS/MSMS/MS

MS vs. MS/MS

GCHPLCCE

Separation Identification

73

CH3 COCH3 CH3 COCH3

Sample Inlet

Sample Inlet

CH3 +COCH3 CH3+COCH3

Ionization& Adsorption

of Excess Energy

Ionization& Adsorption

of Excess EnergyMass AnalysisMass Analysis

CH3 C+OCH3 CH3 C+OCH3

+COCH3+COCH3

+CH3+CH3

+COH+COH

Fragmentation(Dissociation)Fragmentation(Dissociation)

DetectionDetection

Mass Spectrometry

Mass Spectrometry

Collision‐Induced Dissociation

CID (collosion‐induced) or CAD (chemically activated)

Generate

Tandem Mass Spectrometry

• Purpose is to fragment ions from parent ion to  provide structural information

about a molecule

• Allows separation and identification of  compounds in complex mixtures

• Uses two or more mass analyzers/filters  separated by a collision cell filled with Argon or 

Xenon

• Collision cell is where selected ions are sent for  further fragmentation

77

Ion source MS-2MS-1

Mixture of ions

Single ion

Fragments

What is Tandem MS?

• Uses 2 (or more) mass analyzers in a single  instrument

– One purifies the analyte ion from a mixture using  a magnetic field.

– The other analyzes fragments of the analyte ion  for identification and quantification.

78

Applications of Tandem MS

• Biotechnology & Pharmaceutical– To determine chemical structure of drugs and drug 

metabolites.– Detection/quantification of impurities, drugs and their 

metabolites in biological fluids and tissues.– High through‐put drug screening– Analysis of liquid mixtures– Fingerprinting 

• Nutraceuticals/herbal drugs/tracing source of natural products or  drugs

• Clinical testing & Toxicology– Inborn errors of metabolism, cancer, diabetes, various 

poisons, drugs of abuse, etc.

Tandem in Space

80

Tandem in Space MS

Triple Quatrupole

Hybrid InstrumentsESI‐QTOF

Electrospray ionization source + quadrupole mass filter + time‐of‐flight mass analyzer

MALDI‐QTOFMatrix‐assisted laser desorption ionization + quadrupole + time‐of‐flight mass analyzer

Ion‐Source CID with a Single and a Triple  Quadrupole MS

Tandem Mass Spectrometer

TOF

NANOSPRAY TIP

IONSOURCE

HEXAPOLECOLLISIONCELL

HEXAPOLE

HEXAPOLE

QUADRUPOLE

MCPDETECTOR

REFLECTRONSKIMMER

PUSHER

Tandem MS

What is MS/MS?

MS/MS+

+

+ +

+

1 peptide selected for

MS/MS

The masses of all the pieces give an MS/MS spectrum

Peptidemixture

Have only masses to start

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

MS Detectors

• Early detectors used photographic film• Today’s detectors (ion channel and electron

multipliers) produce electronic signals via 2o electronic emission when struck by an ion

• Timing mechanisms integrate these signals with scanning voltages to allow the instrument to report which m/z has struck the detector

• Need constant and regular calibration

Mass Detectors

Electron Multiplier (Dynode)

+e-

primary ion

e-

e- e-L

D

-1000V

-100V

L >> D

Ions are detected with a microchannel plate

MS‐MS Scan Modes

Product ion scans 

• There are two types of product ion scans:

• Full Scan

Product ion are used for qualitative applications to obtain structural information.

• Selected Reaction Monitoring Product ion scans are used for Quantitative target analysis

What are product ion scans? 

• Product ion scans are also know as daughter  ion scans 

• Q1 is set to allow only the transmission of one  m/z 

• The parent ion collides with Argon gas in Q2  to create fragment or product ions 

• Product ions are scanned through Q3

What are precursor ion scans ? 

• Precursor ion scans are also known as parent  ion scans 

• Q3 is set to allow only a fragment ion of one  m/z to pass 

• Q1 is scanned • The precursor ions collide with Argon gas in 

Q2 to create fragment or product ions • Only those compounds which give that 

specific fragment ion are detected 

Precursor ion scans

What are neutral loss scans ? 

• Both Q1 and Q3 are scanned together • Q3 is offset by the neutral loss under 

investigation 

• The precursor ions collide with Argon gas in  Q2 to create fragment ions 

• Only those compounds which give a fragment  having that specific loss are detected

Neutral loss scans

Selectivity of Selected Reaction monitoring  (SRM)

Selected Reaction monitoring (SRM)  Product Ion Scan 

Single Ion Monitoring (SIM)

• Single (or Selected) Ion Monitoring (SIM) is used as a  quantitative scan whereby the molecular ion of the 

analyte is monitored in a narrow amu window. 

• E.g. Orphenadrine may be monitored at 269.9 +/– 0.3 amu (269.6 to 270.2) 

• Other analytes or an internal standard may be  monitored in sequential scans. 

• SIM gives more sensitivity and better selectivity than  monitoring Orphenadrine in full scan MS. 

SIM of Orphenadrine

Protein Sequencing

"Bottom‐Up" Sequencing‐

(most common) a. Cleave protein into peptides.b. Send peptides into MS for sequencing

"Top‐Down" Sequencing‐

(difficult but fast) a. Send intact protein into mass spec.b. Fragment & sequence

Why peptides instead of proteins?1. Increased stability2. Better solubility3. Greater sensitivity4. Easier to sequence if < 20 amino acids5. Fewer (usually <1) translational modifications/peptide5. Cheaper instrumentation (proteins require an FTICR for sequencing)

Breaking Proteins into Peptides

peptides

MPSER……

GTDIMRPAKID

……

HPLCTo MS/MSMPSERGTDIMRPAKID......

protein

MS‐MS & Proteomics

Proteolyzed Proteins Need Separation

Cleaved proteins yield a complex peptide mixture & must be  separated prior to MS.

Separation Characteristics:1. Typically reverse phase (hydrophobicity).

May need multi‐dimensional separation.2. Remove contaminants i.e. detergents, salts3. Reduce complexity but overlapping peaks OK4. Couple directly to ESI/MS

a. Elute in smallest possible volumeb. Peak width of 10‐60 s

Ex: μscale‐

HPLC, capillary electrophoresis, microfluidic chips

Amino Acid Sequencing and  Protein Identification

Breaking Protein into Peptides and Peptides  into Fragment Ions

• Proteases, e.g. trypsin, break protein into peptides.• A Tandem Mass Spectrometer further breaks the 

peptides down into fragment ions

and measures the  mass of each piece.

• Mass Spectrometer accelerates the fragmented ions;  heavier ions accelerate slower than lighter ones.

• Mass Spectrometer measure mass/charge ratio of an ion.

Peptide Fragmentation by MS

• Peptides tend to fragment along the backbone.

• Fragments can also loose neutral chemical groups like  NH3

and H2

O.

H...-HN-CH-CO . . . NH-CH-CO-NH-CH-CO-…OH

Ri-1 Ri Ri+1

H+

Prefix Fragment Suffix Fragment

Collision Induced Dissociation

Peptide Fragmentation

CID fragmentation

b‐ion production

(Michael Kinter and Nicholas E. Sherman, 2000 “Protein sequencing and identification using tandem mass spectrometry”,Wiley, New York)

CIDCID fragmentationfragmentation

yy‐‐ionion productionproduction

Peptide sequencing by MS/MSPeptide sequencing by MS/MS

(Standing 2003 Curr. Opin. Struct. Biol. 13, 595-601)

Protein Identification by Tandem Mass  Spectrometry

SSeeqquueennccee

S#: 1708 RT: 54.47 AV: 1 NL: 5.27E6T: + c d Full ms2 638.00 [ 165.00 - 1925.00]

200 400 600 800 1000 1200 1400 1600 1800 2000m/z

0

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Rel

ativ

e A

bund

ance

850.3

687.3

588.1

851.4425.0

949.4

326.0524.9

589.2

1048.6397.1226.91049.6489.1

629.0

MS/MS instrumentMS/MS instrument

Database search•Sequestde Novo interpretation•Sherenga

DATABASE SEARCH

Police OfficerHeight:  5’7”

Weight:  160 lbs

Gender:  male

Age:  35‐40

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Mass SpectrometristApprox. molecular weight:  30,000

Origin:  bovine liver

Peptide mass list from MS analysis:  

975.4832, 1112.5368, 632.3147, 

803.4134, 764.3892

DATABASE OFKNOWN FELONS

PEPTIDE MASS DATABASEOF KNOWNPROTEINS

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Sequence Tags from Peptide Fragmentation by MS/MS

•

peptide molecular weight (MW)•

partial sequence (region 2)

•

molecular wt before partial sequence (region 1)•

molecular wts after partial sequence (region 3)

A V I/L T

Peptide measured molecular wt = 1927.2

1108.13Partial Sequence- A-V-I/L-T-381.1

region 1 region 2 region 3

One sequence tag includes four components:

1546.11