Glutathionylation and glycation of human hemoglobin

1. Structural proteomics of glutathionylation and
glycation of erythrocyte proteome: A focus on
glutathionylated and glycated hemoglobin.
Monita Muralidharan
Guide: Dr. Amit Kumar Mandal

2. GSH GSSG
GSSG
SH
Hb β +
Cys
S-SG
Hb β
Cys
Clinical marker of
oxidative stress
+ GSH
Oxidative stress
Glutathione
Glutathionylation
• GSHb binds to oxygen 6 fold tighter than the normal hemoglobin (Craescu et.al., JBC,1986).

3. • Modification of N term, K and R side chains
• HbA1c gold standard for accessing glycemic index of a person
•Glycated at the β N-terminus of Hemoglobin
•Under conditions of poorly controlled diabetes there are chances for multiple glycation sites
•Lens crystallin (Bron A J et al., Eye (1993) 7, 260–275)
Glycation

4. Objectives
• Probing structural variations in glutathionyl hemoglobin.
• Characterizing glycated hemoglobin and to study their structural
differences compared to normal hemoglobin.
• To characterize glutathionylated erythrocyte proteins other than
hemoglobin.
• Profiling the glycated erythrocyte proteins apart from hemoglobin.

5. Human hemoglobin structure
β
chain
β chain
α chain
α chain
GS
Objective 1: Probing structural variations in glutathionyl
hemoglobin.

6. Native
Hemoglobin
Mixed
Disulphide
SH
GSH
Glutathionyl
Hemoglobin
Cys
+
2-PDS
S
Cys
S-SG
Cys
HbA
HbA HbA
∆M = 305 Da
MALDI mass spectra

7. • In large excess of D2O, at a fixed pH and temp. H/DX follows pseudo first order kinetics
• Differential solvent accessibility, H-bond strength, inductive and charge effect of
neighboring groups , pH and temperature result in different H/DX rate for different
peptide amide hydrogens

9. MALDI-MS spectra of peptic peptide profile: (A) Hemoglobin; (B) Glutathionyl hemoglobin
Sequence coverage : 30% α globin; 68% β globin
A
B

10. MALDI mass spectra for the peptide β86-102 obtained on hydrogen/deuterium
exchange kinetics.
Hemoglobin Glutathionyl hemoglobin

11. D (t) = (Mt
- M0
) × N …(1)
(M∞
- M0
)
N
D (t) = N - Σ exp – k
i
t
...(2)
і=1
D (t)= N - Ae – k
1
t
- Be – k
2
t
– Ce– k
3
t
…(3)
Initial Rate of H/DX Reaction =kiPi …(4)
N
Σ kiPi
і=1
GSHb
N
Σ kiPi
і=1
HbN
Conformational
Flexibility / Rigidity = ……(5)
The deuterium incorporation at time ‘t’:
0
4
8
12
16
0 25 50 75 100 125
Exchange Time (min)
DeuteriumLevel(Da)
β 86 - 102
GSHb
Hb
(2)
(1)
Deoxy state
•H/DX kinetics of a peptide reflects the conformational dynamics localized in
the region of origin of peptide in the intact protein molecule
Anal. Chem. 2015, 87, 11812−11818

12. Deoxy (T) to Oxy (R) Transition of
HbN

13. 0
4
8
12
16
0 25 50 75 100 125
Exchange Time (min)
DeuteriumLevel(Da)
β 86 - 102
GSHb
Hb
(2)
(1)
Deoxy state
Peptide Mass (m/z) Residues Overall Rate Inference
Compared to HbN
GSHb is more flexible
Covalent binding of
GSH causes this
region to take up an
oxy like conformation
219.98
HbN(d) HbN(o)
β86-102
(ATLSELHCDKL
HVDPEN)
1921.9 (HbN)
2226.9 (GSHb)
-27.73
GSHb(d)
87.89
GSHb(o)
104.4
Craescu et.al., JBC,261(31):14710-6,1986

14. Peptide Mass (m/z) Residues Overall Rate Inference
-1.22
HbN(d) HbN(o)
β (130-146)
YQKVVAGVANALAHKY
H
1868.98
6.2
GSHb(d)
77.8
GSHb(o)
On oxygenation new salt bridges are formed:
βLys132 – βGlu7; βAsn139 – βArg104; and H
bond βHis146-βLys144 is formed
-72.8
•Formation of 3 bonds and
breakage of 2 from
transition of HbNdeoxy to
oxy = Rigidity
•Dramatic change in
flexibillity of Hb on
glutathionylation in deoxy
state
•Transition of GSHb from
deoxy to oxy - Rigidity
Baldwin, J., and Chothia, C, J. Mol. Bio. 129, 175-220.

15. 0
2
4
6
0 25 50 75 100 125
Exchange Time (min)
DeuteriumLevel(Da)
α34 - 46
HbN
GSHb
Peptide Mass (m/z) Residues Overall Rate Inference
GSHb is more flexible when
compared to HbN in both
states in this region of the
molecule
11.9
HbN(d) HbN(o)
α 34 - 46
1585.8
84.4
GSHb(d)
3.7
GSHb(o)
92.6
Oxy HbN: Intersubunit interactions broken :
αLys40 – βHis146
αThr41 – βTyr145
αTyr42 – βAsp99
Deoxy GSHb: α Lys40 – βHis 146; αTyr42 – βAsp99;
αPro44 – βHis97

16. 0
5
10
15
20
25
30
0 25 50 75 100 125
1921.0 (β86-102) 1869.1 (β130-146) 1494.9 (β1-14)
1585.9 (α34-46) 1308.7 (β 32-41) 1635.8 (β115-129)
2910.6 (α1-29) 1799.0 (β15-31) 967.5 (β73-81)
Deuteriumlevel(Da)
Time (mins)
HbN

17. GSHbN
2226.1 (β86-102) 1869.1 (β130-146) 1494.9 (β1-14)
1585.9 (α34-46) 1308.7 (β 32-41) 1635.8 (β115-129)
2910.6 (α1-29) 1799.0 (β15-31) 967.5 (β73-81)
Deuteriumlevel(Da)
Time (mins)

18. Kinetic model prediction of structural transition between
HbN and GSHb
• Significant structural changes observed in the following regions of globin chains
upon glutathionylation: β86−102, β1−14, α34−46, β32−41, β130−146,
β115−129, β73−81.
• In general, glutathionylation caused an increase in the conformational
flexibility of the molecule.
Peptide
Residues
∑N
i{(kiPi)HbNoxy -
(kiPi)HbNdoxy}
Inference
∑Ni{(kiPi)GSHboxy
- (kiPi)GSHbdoxy}
Inference
∑N
i{(kiPi)GSHbdoxy -
(kiPi)HbNdoxy}
Inference
∑N
i{(kiPi)GSHboxy -
(kiPi)HbNoxy}
Inference
(m/z) HbN(d) HbN(o) GSHb(d) GSHb(o) HbN(d) GSHb(d) HbN(o) GSHb(o)
1494.8
β1-14
303.22 Flexible 88.33 Flexible 12.61 Flexible -202.28 Rigid
1308.6
β 32-41
2.24 Flexible 15.02 Flexible 13.29 Flexible 26.08 Flexible
2910.4
α1-29
420.7 Flexible 122.29 Flexible 14.27 Flexible -284.14 Rigid
1798.9
β15-31
218.03 Flexible -35.02 Rigid 129.37 Flexible -123.68 Rigid
967.5 β73-81 28.61 Flexible 122.40 Flexible 18.23 Flexible 112.02 Flexible

19. 10mM NH4

20. 1200 1800 2400 3000 3600 4200 4800
Intensity(%)
0
100
α2βgs2
(+16)
4067.60
α2βgs2
(+17)
3828.33
αβgs
(+11)
2958.45
α
(+9)
1681.68
α
(+9)
1513.73
α
(+8)
1891.65
αβgs
(+12)
2711.88
α
(+7)
2161.80
βgs
(+8)
2022.59
βgs
(+7)
2311.27
αβgs
(+10)
3254.94
α2ββgs
(+17)
3810.23
α2βgs2
(+18)
3615.64
α2βgs2
(+15)
4338.78
m/z
nESI-MS of GSHb tetramer
(25 μM Hemoglobin in 10mM NH4
α2β2 – 64453 Da; α2ββgs – 64758 Da; α2βgs2 – 65063 Da
α globin chain
β globin chain
βgs globin chain
Subunit composition of tetrameric GSHb

21. Determination of solution phase binding affinities of Hemoglobin
T 2D
The dissociation constant (Kd) = [D]2
[T]
[D] and [T] denote the equilibrium concentrations of dimer and tetramer, respectively.
Rsol = [T]
[D]
In solution, the dissociation constant can be calculated as
Kd = [P]0
Rsol(2Rsol+1)
[P]0 - the total protein concentration
RESI-MS = IT
ID

22. nESI-MS of 25 μM HbN tetramer
Kd = 1.8 ± 0.2
nESI-MS of 25 μM GSHb tetramer
Kd = 3.6 ± 0.2
α globin chain
β globin chain
βgs globin chain
Stability of GSHb is perturbed by 2 fold compared to HbN

23. Ion Mobility Separation
Mobility is dependent on factors such as
• Charge
• Size/Shape
o E = uniform electric field
o Ffriction = force of friction (caused by collisions of ions with the buffer gas)
o Fel = force of elimination
o Pbuffer gas = pressure of buffer gas
(http://bowers.chem.ucsb.edu/theory_analysis/ion-mobility/index.shtml)
“Gas Phase Electrophoresis”

24.  The rotationally averaged collision cross-section (CCS) - effective area for the
interaction between an individual ion and the neutral gas through which it is traveling.
The centre of molecule B
comes within the target of
around molecule A so the
two molecules collide.
B
Ar
r
C
B and C
approach A
from this
direction
Collisional Cross Section (σ)

25. (a) Mass spctrum compiled from all ions observed, (b) Plot of drift time versus m/z for GSHb
(a)
(b)

26. Molecules
16+
(Å2
)
17+
(Å2
)
18+
(Å2
)
Average
(Å2
)
SD
(Å2
)
HbN 3593.22 3592.41 3697.20 3627.61 60.27
GSHb-1 3570.70 3551.01 3639.22 3586.98 46.30
GSHb-2 3597.92 3565.56 3637.48 3600.32 36.02
Collisional Cross Section of GSHb
CCS of HbN >
GSHb
Charge state
Collisionalcrosssection(Å2
)
Measure the drift time of the ions (td).
• Calculate drift time (td)
• Calculate td' = td – (c√ (m/z (ion) / 1000) ms
• Correct published cross sections Ω' = (Ω x √ (µ) )/z
• Reduced mass µ = (Mion x Mgas/ / Mion + Mgas)
• Plot td' versus Ω'.

27. Molecule P50
HbN 26.7
GSHb 16.4
GSHb – left shifted curve (P50 – 16.4 mmHg)
Oxygen Dissociation Curve (ODC) for normal hemoglobin (HbN), glutathionyl
hemoglobin (GSHb).
[O2]bound
saturation of Hemoglobin sO2 = ------------- (as each Hb molecule has four O2 biding sites)
4[Hb]total
(1 + 2K2p + 3K2K3p2
+ 4K2K3K4p3
] K1p
= ----------------------------------------------------------
(1 + K1p + K1K2p2
+ K1K2K3p3
+ K1K2K3K4p4
)

28. Sample (HbA1c ≥ 6.5% = Diabetic*)
Boronate Affinity chromatography
Collected and concentrated the glycated pool
Trypsin digestion (E:S = 1:10, 37°C, overnight)
nLC/MS
Database search (PLGS)
0 10 20 30 40 50
min
0
5x103
4x103
3x103
2x103
1x103
Hb N
GHb
mAU
Boronate Affinity Chromatography
*American Diabetes Association, Standards of medical care in diabetes: 2010,
Diabetes Care 33 (Suppl. 1) (2010) S11eS61.
Objective 2: Characterizing glycated hemoglobin and to study their structural differences
compared to normal hemoglobin.

29. NanoLC MSE
profile- Tryptic digest of HbA1c
Fraction
%
0
100
Time
5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00
1
Time
(min)
Flow
Rate
(µL/min)
%A %B
Initial 0.300 97.0 3.0
1.00 0.300 97.0 3.0
61.00 0.300 60.0 40.0
64.00 0.300 15.0 85.0
68.00 0.300 15.0 85.0
70.00 0.300 97.0 3.0
Mobile Phase A: water/0.1% Formic acid
Mobile phase B: ACN/0.1%Formic acid

30. Site of glycation on Hb - β N-terminal peptide (β 1-8)
ΔM = 162 Da

31. Accession Protein Sites
P69905 Hemoglobin subunit alpha V1
, K7
, K11
, K16
, K56
, R92
, K139
, R141
P68871 Hemoglobin subunit beta V1
, K66
, K144
P02042 Hemoglobin subunit delta K17
, K144
P69892 Hemoglobin subunit gamma 2 K95
, K104
, K13
, R144

32. %
0
100
αβ + 1Glc
(+11)
2945.28
αβ
(+11)
2930.55 αβ + 2Glc
(+11)
2960.10
(α2β2) +2G
(+17)
3811.38
(α2β2) +1G
(+17)
3801.81
α2β2
(+17)
3792.27
(α2β2) +2G
(+18)
3599.71
(α2β2) +3G
(+17)
3820.95
(α2β2) +2G
(+16)
4049.55
(α2β2) +1G
(+16)
4039.45
(α2β2) +3G
(+16)
4059.71
25 μM Hemoglobin in 10mM NH4
Glycated Hemoglobin (GHb)
Normal globin subunit
Glycated globin subunit

33. Subunit Stoichiometry of Glycated Hemoglobin

34. Intact globin chain separation of hemoglobin
Deconvoluted mass spectraTotal ion chromatogram
Charge state distribution of normal and glycated globin chains
α globin
β globin

35. Quantification of GHb
α(Glc)
--------------------------------- X 100
α(N) + α(Glc)
GHb-α % =
β(Glc)
--------------------------------- X 100
β(N) + β(Glc) + β(GS)
GHb-β % =
---------------------------------
2
GHb % =
GHb-α % + GHb-β %
Unpaired t test results between HPLC and MS based quantification of HbA1c%
The two-tailed P value = 0.9249
By conventional criteria, this difference is considered to be not statistically significant.
HPLC (HbA1c %) MS (GHb %)
6.75 6.75
17 17.5
21.9 23.3
mins
Absorbance

36. Collisional cross section of GHb
CCS of HbN >
GHb
Charge state
Collisionalcrosssection(Å2
)
Molecule 16+ 17+ 18+ Average SD
HbA 3593.22 3592.41 3697.20 3627.61 60.27
GHb-1 3522.74 3470.42 3534.80 3509.32 34.23
GHb-2 3538.70 3478.71 3555.22 3524.21 40.26

37. Molecule P50
HbN 26.7
GlyHb 33.1
Functional analysis of GHb
GlyHb – right shifted curve (P50 – 33.1
mmHg)

38. HbA1c (%) r (set 1) r (set 2) SD
5 0.113 0.110 0.002
8.6 0.137 0.146 0.006
9.8 0.150 0.152 0.001
10.4 0.162 0.162 0.000
Erythrocyte Membrane fluidity assessment
Ghost RBC
Membrane protein concentration was
calculated just before the experiment
Diluted to required concentration to avoid
depolarization effect due to light scattering
Mix with fluorophore 1,6- diphenyl 1,3,5
hexatriene (DPH)
taken at Ex 360nm; Em 430nm
HbA1c %
Anisotropy(r)
“r” = rigidity

39. Objective 3: Characterize glutathionylated erythrocyte proteins other than
hemoglobin.
Deplete Hemoglobin (Strong cation exchange)
Collect flow though
Concentrate
nLC/MS
Dialyze overnight against 50 mM Ammonium Bicarbonate, pH 7.4

40. Accession Protein Sites Function
P04040 Catalase C392
Protect cells from the toxic effects of hydrogen
peroxide
P32119 Peroxiredoxin 2 C171
Redox regulation of the cell
Q99497 Protein DJ-1 C106
(Active site)
Repairs methylglyoxal- and glyoxal-glycated amino acids
and proteins, and releases repaired proteins and lactate
or glycolate, respectively
P00492
Hypoxanthine guanine
phosphoribosyltransferase
C205
generation of purine nucleotides through the purine
salvage pathway
P60174 Triosephosphate isomerase C72
Involved in gluconeogenesis pathway
P10599 Thioredoxin
C31
(Active site),
C61
Redox reactions through the reversible oxidation of its
active center dithiol to a disulfide and catalyzes dithiol-
disulfide exchange reactions
P09211 Glutathione S-transferase P * C101
Conjugation of reduced glutathione to a wide number
of exogenous and endogenous hydrophobic
electrophiles.
In vitro modified glutathionylated erythrocyte proteins
* Townsend et al., J Biol Chem. 2009 Jan 2; 284(1): 436–445.

41. Objective 4: Characterize glycated erythrocyte proteins other than hemoglobin

42. P04040 CATA HUMAN Catalase OS Homo sapiens GN CAT PE 1 SV 3
R18
, K22
, K97
, R111
, R126
, R129
, R169
, R209
, K242
, R353
, R362
, R379
,
R381
, R443
, R455
, K456
, R457
, K475
, K476
, K503
, R521
, K523
P81605
DCD HUMAN Dermcidin OS Homo sapiens GN DCD PE 1 SV
2 R34
, K38
, R40
, K41
, R43
, K49
, K55
, K56
, K63
, K66
P30043
BLVRB HUMAN Flavin reductase NADPH OS Homo sapiens
GN BLVRB PE 1 SV 3 R45
, R91
, K98
, R123
, R133
, K136
, R173
, K177
P60174
TPIS HUMAN Triosephosphate isomerase OS Homo sapiens
GN TPI1 PE 1 SV 3 K43
,K179
,R32
, R42
, R55
,R172
,R227
P0CG48
UBC HUMAN Polyubiquitin C OS Homo sapiens GN UBC PE
1 SV 3 K27
,K29
, K33
, R42
, K48
, R54
, R604
, R606
P13716HEM2_
HUMAN
Delta aminolevulinic acid dehydratase OS Homo sapiens
GN ALAD PE 1 SV 1 R60
, R66
, R174
, K179
, R190
, R209
, K213
, R221
, R308
P62937
PPIA HUMAN Peptidyl prolyl cis trans isomerase A OS
Homo sapiens GN PPIA PE 1 SV 2 K30
, R36
, K75
, K130
, K132
, R143
, R147
, K150
, K153
, K154
P00441
SODC HUMAN Superoxide dismutase Cu Zn OS Homo
sapiens GN SOD1 PE 1 SV 2 K75
P26447
S10A4 HUMAN Protein S100 A4 OS Homo sapiens GN
S100A4 PE 1 SV 1 K25
,K27
,K47
,K48
, K56
,R39
P32119
PRDX2 HUMAN Peroxiredoxin 2 OS Homo sapiens GN
PRDX2 PE 1 SV 5 R6
, K9
,K15
,K25
,K28
,K33
Q06830PRDX1_
HUMAN Peroxiredoxin 1 OS Homo sapiens GN PRDX1 PE 1 SV 1 K6
,K36
,R109
, R127
, K119
,K196
,K198
P30041
PRDX6 HUMAN Peroxiredoxin 6 OS Homo sapiens GN
PRDX6 PE 1 SV 3 K55
,K62
, R63
, R105
, R107
, K121
, K124
, R131
, R173
, K198
,K214
,K215
, R218
Q13228
SBP1 HUMAN Selenium binding protein 1 OS Homo
sapiens GN SELENBP1 PE 1 SV 2 R381
, K396
In vivo modified glycated erythrocyte proteins from 5-16% HbA1c samples

43. P00352
AL1A1 HUMAN Retinal dehydrogenase 1 OS Homo sapiens GN
ALDH1A1 PE 1 SV 2 K36
,K64
, R67
, R77
, R97
, R321
,R325
,R394
, K409
, K418
P04075
ALDOA HUMAN Fructose bisphosphate aldolase A OS Homo
sapiens GN ALDOA PE 1 SV 2 R42
, R55
, K316
, K317
, K321
P02768
ALBU HUMAN Serum albumin OS Homo sapiens GN ALB PE 1
SV 2 R2
,K139
, R188
, K197,
K201
,R211
,R220
,R474
P00338
LDHA HUMAN L lactate dehydrogenase A chain OS Homo
sapiens GN LDHA PE 1 SV 2 R156
, R314
, K317
, K327
P06703
S10A6 HUMAN Protein S100 A6 OS Homo sapiens GN S100A6
PE 1 SV 1 K26
, R55
, R62
, K89
P15531
NDKA HUMAN Nucleoside diphosphate kinase A OS Homo
sapiens GN NME1 PE 1 SV 1 R26
, K30
P50395
GDIB HUMAN Rab GDP dissociation inhibitor beta OS Homo
sapiens GN GDI2 PE 1 SV 2 R68
, R98
, K112
,K164
P67775
PP2AA HUMAN Serine threonine protein phosphatase 2A
catalytic subunit alpha isoform OS Homo sapiens K29
,R115
,R302
P00492
HPRT HUMAN Hypoxanthine guanine
phosphoribosyltransferase OS Homo sapiens GN HPRT1 PE 1
SV 2 R47
, R50
P11142
HSP7C HUMAN Heat shock cognate 71 kDa protein OS Homo
sapiens GN HSPA8 PE 1 SV 1 R508
, K530
, K250
, K256,
R257
, K499
, R508
, K549
P40925MDHC_H
UMAN
Malate dehydrogenase cytoplasmic OS Homo sapiens GN
MDH1 PE 1 SV 4
K78
, R91
, K102
, K109
, K117
, K120
, K121
, K148
, R161
, R229
, R237
, K238
, K247
,
K297
, R309
, K317
P52209
6PGD HUMAN 6 phosphogluconate dehydrogenase
decarboxylating OS Homo sapiens GN PGD PE 1 SV 3 K50
, K58
, R254
, K260

44. P63208
SKP1 HUMAN S phase kinase associated protein 1 OS
Homo sapiens GN SKP1 PE 1 SV 2 R135
, K136
P04075
ALDOA HUMAN Fructose bisphosphate aldolase A OS
Homo sapiens GN ALDOA PE 1 SV 2 R42
, R55
, K316
, K317
, K321
P00918
CAH2 HUMAN Carbonic anhydrase 2 OS Homo sapiens
GN CA2 PE 1 SV 2 K8
, K23
, R26
, K79
, K112
, K170
, R244
, R252
, K255
P00491
PNPH HUMAN Purine nucleoside phosphorylase OS
Homo sapiens GN PNP PE 1 SV 2 R24
,R58
,R173
Q9NRV9
HEBP1 HUMAN Heme binding protein 1 OS Homo sapiens
GN HEBP1 PE 1 SV 1 R56
, K64
, R125
Q15257
PTPA HUMAN Serine threonine protein phosphatase 2A
activator OS Homo sapiens GN PPP2R4 PE 1 SV 3 ,
R221
, R227
, K228
, R327
Q9NZD4
AHSP HUMAN Alpha hemoglobin stabilizing protein OS
Homo sapiens GN AHSP PE 1 SV 1 R63
P06733
ENOA HUMAN Alpha enolase OS Homo sapiens GN ENO1
PE 1 SV 2 R252
P00915
CAH1 HUMAN Carbonic anhydrase 1 OS Homo sapiens
GN CA1 PE 1 SV 2 K45
, K80
, K149
, R246
, R254
P07195LDHB_
HUMAN
L lactate dehydrogenase B chain OS Homo sapiens GN
LDHB PE 1 SV 2 K81
, R157
, R169
, R171
, K309
, K317
, K318
, K328
, K331
P20810
ICAL HUMAN Calpastatin OS Homo sapiens GN CAST PE 1
SV 4 K37
, K38
, R86
, R310
, K452
, K457
, K461
, R604
, K684
, K687
, K691
, K693

45. Conclusion
• GSHb is much more flexible than HbN; Oxygen dissociation curve shows high
oxygen affinity for GSHb compared to its normal counterpart;
• Glycation leads to an over all decrease in the collisional cross section of HbN
making it more compact.
• Probable candidates that experience glycation and glutathionylation and
characterized their site of modifications. These modifications could lead to
functional changes in proteins and may be associated with several disorders of
oxidative stress as well as diabetes

46. PUBLICATIONS
1. Structural perturbation of human hemoglobin on glutathionylation probed by hydrogen-deuterium
exchange and MALDI mass spectrometry.
Mitra G#
, Muralidharan M#
, Pinto J, Srinivasan K, Mandal AK.
# Authors contribute equally
Bioconjug Chem. 2011 Apr 20;22(4):785-93.
2. Glutathionylation Induced Structural Changes in Oxy Human Hemoglobin Analyzed by Backbone Amide
Hydrogen/Deuterium Exchange and MALDI-Mass Spectrometry.
Mitra G#
, Muralidharan M#
, Narayanan S, Pinto J, Srinivasan K, Mandal AK.
# Authors contribute equally
Bioconjug Chem. 2012 Dec 19;23(12):2344-53
3. Protein Structure-Function Correlation in Living Human Red Blood Cells Probed by Isotope Exchange-based
Mass Spectrometry.
Sreekala Narayanan #
,Gopa Mitra#
, Monita Muralidharan, Boby Mathew, Amit Kumar Mandal
# Authors contribute equally
Analytical Chemistry 11/2015; 87(23).
4. Mass spectrometry based characterization of Hb Beckman variant in a falsely elevated HbA1c sample
Rajdeep Das, Monita Muralidharan, Gopa Mitra, Vijay Bhat, Boby Mathew , Debnath Pal, Cecil Ross, Amit
Kumar Mandal

47. Acknowledgements
Clinical Proteomics Unit, Division of Molecular Medicine,
SJRI
Funding Agency:
CSIR, Govt. of India

48. Thank you!

49.  By adjusting the quadrupole rf parameters
achieve a transmission profile that is relatively
uniform between m/z 300 and 4500.

59. Protein Sites
Hemoglobin subunit alpha *V1
, K7
, K11
, K16
, K56
, R92
, K139
, R141
Hemoglobin subunit beta *V1
, *K66
, K144