Micromass Quattro Ultimatriple quadrupole mass spectrometric detector

HPLC system (LC)

Electrospray ionisation source (-ve & +ve ion)

Photodiode array detector (PDA)

Fluorescencedetector

The Equipment LC-MS/MS

A little bit about triple quadrupole mass spectrometry.

Hexapoleion bridge

(I) (II)

Z-sprayion source

Extractioncone

Quadrupole(MS1)

Quadrupole(MS2)

Pre-filterPost-filter

Hexapolecollision cell

Photomultiplier

Phosphor

Ion drag

Focus ring

Conversiondynode

Whisperdetector

Electrospray or APcI ion

source

TM

A little bit about electrospray ionisation.

Desolvatongas manifold

Isolation valve

Hexapoleion bridge

Extractioncone

Ionblock

VentCleanablebaffle

Samplingcone

Measurement of protein mass by triple quadrupole MS.

Protein (pure) sample(prepared by investigator)

■ Multiple-charged ion series - deconvolution gives molecular masses

■ Intractable analysis for complex protein mixtures. Limited mass resolution.

Mass spectrometerSample infusion

Infusion pump

Electrospraysource

Parent ions

Proteinn+

Protein(n+x)+

Mass analyser-2

OFF

Photomultiplier detector

Response

020406080

100

600 1100Molecular mass (Da

(%)

Mass analyser-1

Collisioncell

Applications:Peptide mapping of haemoglobin modified by methylglyoxal

MGmin-Hb

0

20

40

60

80

100

15000 15250 15500 15750 16000 16250 16500Molecular mass (Da)

Res

pons

e (%

)

15129 (a -chain)

15870 (b -chain)

0

20

40

60

80

100

500 700 900 1100 1300 1500 1700Molecular mass (Da

(%)

Control haemoglobin

0

20

40

60

80

100

15000 15250 15500 15750 16000 16250 16500Molecular mass (Da)

Res

pons

e (%

)

15129 (a -chain)

15870 (b -chain)

Detection of protein biomarkers by LC-MS/MS:Multiple reaction monitoring (MRM)

HPLC

Mass analyser-1

Parent ion

Electrospraysource

Biomarker+

Mass analyser-2

Biomarker fragment

Collision cell

Fragment ion

+

■ High specificity

■ (LC, MS1 and MS2 resolution)

■ High sensitivity

■ Biomolecule compatible

Mass spectrometer

Enzymatic hydrolysate(prepared by investigator)

Photomultiplier detector

Response

■ Biomarker screening in 75 min per sample.

Advanced glycation endproducts

HCCO

NHCHCO

NHN N (CH2)4(CH2)4

Bis(lysyl)imidazolium crosslinks

MOLDGOLD

+HCCO

NHCHCO

NHN N (CH2)4

CH3

(CH2)4+ +

DOLD

CHCO

NHHC

CO

NHN N (CH2)4(CH2)4

H2C(CHOH)2CH2OH

ArgpyrimidinePentosidine crosslink

+

CHCO

NH

HCCO

NH(CH2)3 NH

N

HN N(CH2)4 N

NCH3

CH3

OHNHHCCO

NH(CH2)3

Ne-Carboxymethyl-lysine (CML) Ne-Carboxyethyl-lysine (CEL)

Monolysyl adducts

HCCO

NHNHCH2CO2

-(CH2)4

HCCO

NHNH(CH2)4 C

CH3

CO2-

H

Pyrraline

HCCO

NH(CH2)4 N

HOCH2

HO

AGEs with intrinsic fluorescence

Hydroimidazolones

G-H1 MG-H1

HCCO

NH

HN

NNH(CH2)3

H

H

O

HCCO

NH

HN

NNH

CH3

H

O

(CH2)3HN

NNH

CH2

H

O

HOCH2(CHOH)2

HCCO

NH(CH2)3

3DG-H1

LC-MS/MSwith stable isotope-substituted

internal standards

Detection of protein biomarkers by LC-MS/MS: Calibration, sample de-lipidification, ultrafiltration & enzymatic hydrolysis

Delipidification and AGE fractionation■ Ultrafiltration to separate protein AGE residues and free AGEs■ Ether or methanol/chloroform extraction

Analytical performance■ Limits of detection: 20 – 500 fmol.■ Recoveries: >80%; 94-100% for amino acids■ Interbatch c.v.: <10% (n = 6)

Enzymatic digestion:■ Pepsin (+ thymol)■ Pronase E (under nitrogen, penicillin and streptomycin added)■ Prolidase and aminopeptidase (under nitrogen)

Internal standardisation and calibration■ Standards and stable isotope-substituted standards

e.g. CML and [13C6]CML, MG-H1 and [15N2]MG-H1

Detection of protein biomarkers by LC-MS/MS:Retention of amino acids and AGEs and use of column switching

Hypercarb graphitic columns retain underivatised amino acids, allowing for diversion of non-volatile salts to waste.

Non-volatile salts to waste

Hypercarb column (2.1 x 250 mm)

Hypercarb column

(2.1 x 50 mm)

SampleTo

MS/MS

Column switching facilitates elution of hydrophobic analytes and column washing.

Switching valve

Examples of detection by multiple reaction monitoring (MRM): CML

N-Carboxymethyl-lysine (CML)

CO2HHC

NH3+

NH CH2 CO2HCO2H

HCNH3

+

Fragment ionMr = 130.1

CML204.9

CML detected in plasma protein of a normal healthy human control subject.

0

20000

40000

60000

5 6 7 8 9Retention time (min)

Dete

ctor

resp

onse

(c

ount

s)

CMLMRM: 204.9 > 130.1

0

20000

40000

60000

5 6 7 8 9Retention time (min)

Dete

ctor

resp

onse

(cou

nts)

[13C6]CMLMRM: 210.9 > 136.1

Examples of detection multiple reaction monitoring (MRM): Methylglyoxal-derived hydroimidazolone

Methylglyoxal hydroimidazolone (MG-H1)

CO2HHC

NH3+

NHN

NH CH3

O

H NH3N

NH CH3

O

H+

MG-H1Mr = 229.2

Mr = 114.3

MG-H1 detected in rat retinal protein hydrolysate of a STZ diabetic rat.

0

50000

100000

150000

22 23 24 25 26 27Retention time (min)

Dete

ctor

resp

onse

(cou

nts)

MG-H1MRM: 229.2 > 114.3

0

50000

100000

150000

22 23 24 25 26 27Retention time (min)

Dete

ctor

resp

onse

(cou

nts)

[15N2]MG-H1MRM: 231.2 > 116.3

Arg 14.2 175.2 70.3 15 H2CO2, NH2C(=NH)NH2

Lys 6.0 147.1 84.3 15 H2CO2, NH3

Met 9.2 150.0 104.2 11 H2CO2

MetSO 7.5 166.1 102.2 14 CH3SOH

CML 7.4 204.9 130.1 12 NH2CH2CO2H

MG-H 23.7 229.2 114.3 14 NH2CH(CO2H)CH2CH=CH2

Pent 16.5 379.3 250.4 22 NH2CH(CO2H)CH2CH2CH=CH2

Analyte Rt Parent Ion Fragment ion CE Natural Fragment loss (min) (Da) (Da) (eV)

Mass spectrometric multiple reaction monitoring detection of protein biomarkers

Peptide mapping to identify sites of protein modification

Mass spectrometer

Mass analyser-1

Collisioncell

Electrospraysource

Parent ionsPeptides

+

Tryptic digest of protein sample(prepared by investigator)

HPLC PDA

Resolution of peptide fragments by LC

Mass analyser-2

OFF

Photomultiplier detector

0

50

100

0 10 20 30 40

SIR (%

)

Single ion response for each peptide

Peptide map

Biolynx match of peptide M+ with theoretical digest.Locate modified peptide M+ ion

Peptide mapping to identify glycation sites.

Glycation of human serum albumin by methylglyoxalLocation of glycation sites by LC-MS peptide mapping

MS detection of peptide fragments by LC-MS and quantitation of the MS response Peptides are partially resolved by HPLC with ODS

chromatography and detected by positive ion electrospray MS.

Limited proteolysis of MGmin-HSA and HSA control Reduction of disulphide bonds with dithiothreitol. S-Alkylation of cysteine thiols by iodoacetamide. Digestion with trypsin (and independently with Glu-C for

corroboration).

0

50

100

150

0 10 20 30 40 50 60

Retention time (min)

Resp

onse

(cou

nts

x 10

9)

MS detection of peptide fragments by LC-MS and quantitation of the MS response

Peptide responses are normalised to the C-terminal peptide (LVAASQAALGL).

Loss of peptides in MGmin-HSA digest was quantified by the mean normalised peptide response for MGmin-HSA, relative to HSA control (mean c.v. = 11%). This is assumed due to glycation

The glycated peptides were also detected as modified dipeptides (resistant to proteolysis in tryptic maps).

02468

1012

30 32 34 36 38 40Retention time (min)

Norm

alis

ed.

resp

onse

HSA

MGmin-HSA

Modification of arg-410

Peptide(T52) FQNALLVR

02468

32 34 36 38 40 42Retention time (min)

Norm

alis

ed

.re

spon

se

MGmin-HSA

HSA

LC-MS/MS peptide mapping can also be used to locate glycation, oxidation and nitration markersLocation of MG-H1 residues in human serum albumin modified minimally by methylglyoxal

02468

1012

30 32 34 36 38 40Retention time (min)

Norm

alis

ed.

resp

onse

HSA

MGmin-HSA

Ion chromatograms for peptide T52 (containing R410)

0

2

4

6

8

32 34 36 38 40 42

Retention time (min)

Norm

alis

ed .

resp

onse

MGmin-HSA

HSA

Ion chromatograms for dipeptide T52-53 (containing MG-H1-410)

010203040

1350 1370 1390 1410 1430 1450

Mass (Da)

Resp

onse

(c

ount

s)

1406.8 Da

Predicted mass of T52-53 FQNALLVRMG-H1YTK 1406.9; found peptide mass 1406.8 Da.

Glycation of human serum albumin by methylglyoxalLocation of glycation sites by LC-MS peptide mapping

R410R218

R114R186 R428 Arg MG-H1

(mol%)114 36186 25218 31

410 89428 25

Modification hotspot: Arg-410 Drug binding site 2. Active site of

esterase activity.