Transcript Lecture Note 3

IonSpec
FT ICR Isotopic Resolution of Proteins: Myoglobin
12 T qFT ICR MS (UWO)
MS/MS with FT ICR
1. SORI: Sustained off-resonance irradiation
Excitation of selected ions slightly off- resonance. This results in ions
being alternatively accelerated and decelerated limiting the cyclotron
radius. Collision gas is introduced briefly to produce collision
activated dissociation (CAD). The fragment ions are then produced
close to the center and can be detected repetitively to give better
sensitivity and resolution.
Fragmentation of peptides gives mostly b an y ions (as in CAD in quad)
2. IRMPD: Infrared multi-photon dissociation
External IR laser provides energy to activate bonds and
cause dissociation. Does not require introduction of gas pulses
and therefore shortens the duty cycle
Fragmentation of peptides gives mostly b an y ions (as in CAD in quad)
3. ECD: Electron Capture Dissociation
Heated filament inside the cell emits electrons that are capture by
the ions inside the cell. The precursor ion must be doubly or more
charged to yield singly charged radical ion. Dissociation or
fragmentation will occur a the amide bond between the NH and CH (N-Ca)
giving primarily c and z ions. It is therefore complementary to SORI
IRMPD and classical CAD in quad collision cell. Another advantage is
for the analysis of PTMs since the amide bond is cleaved first not the
phospho bond or glycosidic linkage.
H2N
H
C
+ OH
C
O
H
N
R1
H
C
H
N
C
+
e-
OH H
C N
H
C
H2N
.
R1
R2
O
H
C
H
N
C
OH H
C N
H
C
H2N
O
+
z
c
y4
x4
z4
O
H2N
H
C
C
y3
H
N
H
C
C
c1
a2
x2
y2
z2
O
H
N
R2
b1
z3
O
R1
a1
x3
H
C
C
c2
a3
z1
O
O
H
N
R3
b2
y1
x1
H
C
C
H
N
b3
c3
a4
H
C
R5
R4
b4
c4
.
R2
R1
R2
H
C
a5
C
OH
C
H
N
J
L
C
Complementary Sequencing from Two Different MS/MS Techniques
4+
712.18
100
2+
81.97
Electron capture dissociation (ECD) → c, z ions
80
Infrared multiphoton dissociation (IRMPD) → b, y ions
3+
906.47
3+
949.11
60
2+
1216.97
969.15
2+
1415.28
1721.44
2+
897.51
40
2+
1159.99
2+
1104.02
656.23
528.21
2+
811.66
4+
712.43
2+
1075.06 1127.03
3+
863.83
2+
998.55
1033.99
1656.25
20
2+
755.17
1336.88
838.64
414.15
2+
948.03
612.24
2+
868.51
493.31
3+
541.67
2+
1104.61
1925.30
1525.88
1657.78
2+
1054.58
594.37 642.46
1830.70
2+
1358.84
2+
989.07
1187.66
1527.08
1272.63
1832.43
1927.20
0
400
600
800
1000
1200
m/z
1400
1600
1800
2000
R. Zubarev, N. L. Kelleher, and F. W. McLafferty JACS 1998 120 (13) 3265
“Top-Down” Sequencing
Inject intact protein in the FT ICR and perform one or more (or combine)
fragmentation method (ie ECD and ECD + IRMPD).
Mol. Cell. Proteomics 2003 2: 1253-1260
Advantages of Top-Down Sequencing
Can get the identity of the proteins from full sequence including the
post-translational modifications which are often missed by
bottom-up approaches since coverage tends to be poor 5-30%.
However:
Size limitation ~45kDa
Limit of detection: larger proteins are more difficult to ionize
Hybrid FTICR instruments with quadrupole for ion selection and CAD
before entering the ICR cell: qFTMS (Bruker, IonSpec) and
linear ion trap (LTQ FT, Thermo Finnigan); much more sensitive.
Orbitrap
Latest on the market with a new type of mass analyzer:
Hybrid instrument combining with a linear trap with the orbitrap
FT MS
Image current detector
High resolution
Accurate mass
FT MS with no magnet!
Orbitrap
2 Detectors in one instrument
FT MS with no magnet!
Detection and Measurement of Ions in the Orbitrap
The orbitrap mass analyzer employs the trapping of pulsed ion beams
in an electrostatic quadro-logarithmic field. This field is created
between an axial central electrode and a coaxial outer electrode. Stable
ion trajectories combine rotation around the central electrode with
harmonic oscillations along it. The frequencies of axial oscillations and
hence mass-to-charge ratios of ions are obtained using fast Fourier
transform of the image current detected on the two split halves of the
outer electrode.
Hardman M, Makarov AA. Anal. Chem. 2003 Apr 1;75(7):1699-705.
Orbitrap: summary
Important new tool in “bottom-up” proteomics
Excellent mass accuracy especially with internal standard < 1.0 ppm*:
mass accuracy is critical for precursor ion
Can perform MS/MS in linear trap and fragments analyzed i n either the
Linear trap (faster but lower resolution) or in the Orbitrap.
Disadvantages:
Poor for intact proteins
Cannot do MS/MS by IRMPD, ECD, etc.
* M. Mann et al. Mol. Cell. Proteomics 2005, 2010
Quantitation by MS
LC MS is not strictly quantitative: different peptides ionize differently
in both MALDI and ESI: Need internal calibrants most often with isotope
enriched similar chemical species
-Use isotopes enrichment:
- ICAT, cleavable ICAT, iTRAQ
- Labeling with N15, C13
- Labeling with O18 (With protease digestion)
- Labeled amino acid added to growth media (SILAC)
Leu (d3), Ser, Tyr, Lys (d4), Arg (13C, 15N)
- Internal calibrant with isotoptically labeled peptide
(AQUA)
-Others:
1. add known amount of protein to mixture and digested and/ or
normalized against proteins that do not change in intensity
2. Intensity of signal (normalized)
Quantitative Proteomic Profiling
Stable Isotope labeling
2D GE
In vitro labeling
In vivo labeling
N14/N15 media
SILAC
Intensity-based quantitation
N-terminal
peptide labeling
C-terminal
peptide lableling
Amino AcidBased labeling
Cys:ICAT
iTRAQ
Esterification
(CD3OH)
16O/18O
incorporation
via proteolysis
ICAT: Isotope Encoded Affinity Tags
To quantitate expression of proteins from cells exposed to different
conditions by ESMS (ion trap) and avoid problematic 2D gels:
Add an affinity label- biotin (avidin tight binding) linked by a short
spacer which contain either 0 or 8 deuteriums and alkylating portion
to react with SH of cysteines.
Affinity chromatography: retains only cysteines labeled peptides
Measure relative intensities of peaks separated by 8 Da.
Peptides only differ by deuterium and separate and ionize the same.
Improvement in separation affinity column (30 fractions) each
ion exchange (30 fractions) and RP HPLC (30 fractions)
2700 samples to analyze by ESMS !!
Original ICAT Reagent
Light ICAT d0 , X = H
O
Heavy ICAT d8, X = D
NH
HN
HS-Cys-peptide
X X
NH
S
O
Biotin Affinity tag
O
X
O
X
Linker with labels
Gygi et al, Nature Biotech (1999) 17 994
Aebersold et al, Curr. Opin. Biotech (2003) 14, 110
O
X
X
X
X
H
N
I
O
Reactive group
ICAT Procedure
1. Denature (urea +SDS) and reduce proteins (TCEP)
(Tris- caboxyethylphophine) HCl
2. Incubate with ICAT reagent
3. Combine control and test samples
4. Digest with trypsin
5. Chromatography:
A. Affinity Biotin/Avidin:
rejects non labelled peptides, simplifying mixture
B. Reverse Phase HPLC
7. MS to measure relative intensity of peaks differing
by 8 Da and MS/MS to ID peptides
Denature and reduce
SH
HS SH
HS
SH
HS SH
SH
SH
SH
HS
HS SH
SH
HS SH
Labeling with heavy ICAT
Labeling with light ICAT
S S
S
S
S
S
S S
S
S
S S
S
S S
S
Mix and Trypsin digestion
S
S
S
S
S
S
Non retained
Affinity capture biotin/avidin
Peptides
S
S
S
S
S
S
LC MS
Ratio
Light /heavy
MS/MS
ID
ICAT (cont..)
L
S
8 Da
H
Select doubly
charged ion
S
(281.5)
MS/MS
560 568
m/z
m/z
LC MS
Ratio ~ 60:40
Database Search
(MASCOT)
Identification of protein
ICAT Isotope encoded affinity tags
Limitations:
1) does not work with proteins with no cysteine
(in yeast 20%, more in other organisms)
2) only identify small peptide portion of these proteins
ie miss post-translational modifications
3) limited MS/MS sequencing because interference
of the ICAT label
4) Long deuterated PEG chain changes HPLC
retention time. The intensity in the MS is not accurate
to quantitate both species (ie arrive at different times
at the MS).
Solid Phase ICAT
Isotope tag X = H or D (Dm = 7)
h cleavage
CX3
CX
H
N
CH3
NO2
HS-Cys-peptide
O
O
O
N
H
X3C
N
H
OCH3
I
Thiol reactive
O
Photocleavable linker
Glass beads
Better, but still problems: reagent is very light sensitive (and expensive)
Zhou et al., Nature Biotech. 2002, 19, 512
Quantitation: Labeling with O18
Procedure:
On one test sample extract proteins, reduce, alkylate
cysteines and treated with trypsin in the presence of H2O18 : get
incorporation of O18 at the C terminal carboxylic acid (from attack
of H2O18 on the acyl-enzyme intermediate
Mix this digest with tryptic digests the control samples
Performed in the presence of H2O. Perform MS and
measure relative intensity of peaks differing by 2 Da.
One can get incorporation of 2 O18 simplifying the analysis by
promoting trypsin catalyzed second exchange. Works better with
Arg than for Lys
Quantitation: Labeling with O18
Get 1 (or 2 O18)
Mass difference of 2 (or 4): need
high resolution MS !
MS of tryptic peptides digest labeled with O18 vs control
18
O
100%
C
O
Intensity
With Arg can get exclusively + 4 Da
With Lys get a mixture of + 2 and + 4 Da
C
OH
OH
Arg (Lys)
18
O
Arg (Lys)
C
18
OH
Arg (Lys)
+2 +4
m/z
Ratio labeled vs unlabeled ~2:1
Quantitation: Labeled Amino Acids
SILAC (stable isotope labeling with amino acids
In cell culture)
Labeled amino acid added to growth media
Leu(d10), Ser (d3), Tyr (d2), Lys (d4), Arg (N14, C13)
With auxotrophs strains or with media depleted
in these amino acids
Limitations:
Some Scrambling
Not generally applicable to all types of experimtns
(eg Human tissues)
J. Proteome Research 2002,1 , 345-350
Rapid Comm. Mass Spec. 2002,16 2115-2123
SILAC Procedure
Leu-d3
Incorporation of Leu-d3 in proteins at various time points
Ong, S.-E. (2002)
Mol. Cell. Proteomics 1: 376-386
The use of dialyzed serum avoids nonspecific incorporation
of non-labeled leucine derived from serum in the Leu-d3 samples
Ong, S.-E. (2002)
Mol. Cell. Proteomics 1: 376-386
Quantitation of nine proteins during C2C12 cell differentiation by SILAC
Ong, S.-E. (2002)
Mol. Cell. Proteomics 1: 376-386
Observed ratios are similar to expected ratios in mixing experiments
Ong, S.-E. (2002)
Mol. Cell. Proteomics 1: 376-386
Labeling with 15N or 13C
Each peptide is enriched
with 15N and/or 13C
Gives complex mixture
difficult to interpret
Most useful in MS only,
with FT ICR MS and
“accurate mass tag”
Quantitation in the MS/MS: the iTRAQ approach
iTRAQ: Isotope Tags Reagents for Accurate Quantitation
Quantitation for drugs and metabolites are usually done in the MS/MS mode:
to minimize chemical noise and get better accuracy.
Design sets of chemicals tags to react with N-terminal amines (and Lysine)
that would have the same retention times on LC AND the same mass (isobaric)
in the MS experiments but for which fragments (reporter) would differ in the
MS/MS.
Isobaric tag mass = 142
Reporter group
114,115,116.117
NH -peptide
Balance group
31,30,29,28
MS/MS Fragmentation site
The Four iTRAQ Reagents
CH 3
CH3
CH 3
N
N
N
N
*
Y
Y= leaving group
H2DC
N
N
O
H2DC
O
*31
114
115
CD2H
N
N
N
116
*
O
Y
29
H2DC
N
*
30
CDH2
N
Y
N
Y
O
117
28
N
O
N
N
H
N
*
OH
O
R
*
CID
O
N
*C
N
N
HN
OH
+
O
*
Peptide
Fragments
R
Quantifying Fragment
m/z 114
32 Da
Peptide Identifciation
iTRAQ: Procedure
Up to 4
test samples
ITRAQ: Advantages and Disadvantages
Advantages
-Can perform up to four quantitation experiment at once.
Allow for time course experiments e.g. 0 min, 10 min, 30 min, 60 min
each with a different label
-The reporter group does not interfere with the peptide fragmentation
so that quantitation and peptide ID can be done in the same experiment.
Disadvantages
-React with other amine groups ie lysine side chain and amines in
buffer. The amine containing buffers have to be replaced before
labeling.
- Very expensive reagents!
Post-translational modifications (PTMs)
Modifications are very important sometime determinant for function
(over 200 known modifications)
Most important:
Phosphorylation
pTyr,
pSer, pThr
Glycosylation
N-linked
O-Linked
Acetylation
Methylation
Acylation
Farnesyl
Myristoyl
Sulfation
Disulfide
Deamidation
Ubiquitination
Nitration
Truncation
+80
Enzyme Activity, signaling
+++
+/++
Cell/cell recognition
>800
>203
+42
+14
++
++
+++
+++
+204
+210
+++
+++
-2
+1
>1000
+45
-x
+80
++
+++
+++
++
+++
Protein stability
Gene expression
Cellular Localization
Protein/protein interactions
Protein stability
Protein/protein interactions, aging
Protein degradation
Oxidative damage
Detection of Phosphopeptides at Low Levels
Phosphorylation: > 100,000 possible sites in human; ~2000 known
~1/3 of human proteins are phosphorylated
Notoriously difficult to analyze, especially at low levels.
sub-stoichiometry
poor ionization and ion suppression
other factors? absorption on LC columns
Multiply phosphorylated are not observed
What factors are responsible for the absorption of phosphopeptides,
especially mutliply-phosphorylated peptides on LC?
Complexation of phosphate groups with free Si-OH on
C18 RP material?
Smith R. D. et al (2004) J. Mass Spectrom. 39:208-215
Detection of Phosphopeptides at Low Levels
Several approaches for enrichment have been described:
• IMAC with Fe+3 (or Ga+3, Al+3) and variations
followed by LC MS
•Beta-elimination/addition of thiols
• Graphite columns to retain the
“very hydrophilic phosphopeptides”
• Addition of H3PO4 to the sample to elute multiply-phosphorylated
peptides bound to silica on the RP column*
•TiO2 columns for enrichment
* Smith R. D. et al (2004) J. Mass Spectrom. 39:208-215
Post-translational modifications (PTMs): Phosphorylation
Phosphorylation: 100,000 possible sites in human; ~2000 known
~1/3 of human proteins are phosphorylated.
One of the main challenge in proteomics
pTyr: (~1%)
Chemically stable: observed +80
enrichment with phospho Tyr specific antibodies
or with IMAC (immobilized metal affinity chromatography)
pThr: (~10%)
-less stable but can be observed in MS
pSer: (~90%)
Unstable: usually undergoes by base or in MS b-elimination to
dehydro-Ala (-98) which can react with nucleophile (thiols, amines)
enrichment by IMAC
D. Kalume et al Curr. Opinion in Chem. Biol. 2003, 7:64-69
Immobilized Metal Affinity Chromatography: IMAC
Based on affinity of phosphates to certain metals Fe+3,Ga+3
Phosphopeptides are preferentially retained
but so are strongly acidic peptides (Asp, Glu)
Solution: esterify with MeOH/ HCl prior to column
Phosphorylation can be verified by alkaline phosphatase
Lost of 80 Da by MS
Note: Phosphopeptides tend to inonize less efficiently
than their non-phosphorylated counterpart
IMAC
OH
O
O
C
CH3
CH
C
OH
O
H2C
O
M
N
H2C
O
C
O
NH
O
P
O
O
HN
O
H2C
CH
C
HO
M = Fe , Ga
CH
C
O OH
P
C
H2
O
O
Phosphopeptides: after IMAC
Modification: esterification of carboxylates improves sensitivity
Nat. Biotech. 2002, 20, 301
Location of Phosphorylated Residue(s) by MS/MS
Select precursor ion (+80) for MS/MS
PhosphoTyrosine:
The phosphate is stable : look for difference of 243 in between the y (or
b) ions (Tyr 163 + Phosphate 80)
PhosphoSerine: The phosphate is very labile and is lost in the collision of the
MS/MS experiment resulting in a dehydroalanine residue: the peptide bond is
then fragmented. The location can be determined by the difference of 69
between 2 y adjacent (or b) ions
PhosphoThreonine: The phosphate is partially lost followed by peptide bond
fragmentation. The location of 83 between adjacent y (or b) ions. This is most
often accompanied by series of ions containing intact phosphothreonine.
Therefore the location can be determined by mass difference of 181 (Thr 101 +
80)
Phosphoserine and b-elimination
Base :
MS/MS
b-Elimination
OPO3H2
NH
H
NH
NH
O
-69
NH
O
R
-98
Ba(OH)2
R
+ H3PO4
Dehydroalanine
OH
O
H
-87
NH
NH
O
R
Serine
Phosphate group of serine is very labile and can be b-eliminated by
base treatment OR in the MS experiment
MS/MS of Phosphoserine vs Serine
OH
NH
O
NH
O
dehydroalanine
R
H
NH
NH
O
+ H3PO4
R
serine
yx
y*x+1
69
m/z
yx
yx+1
87
m/z
MS/MS of PhosphoThreonine vs Threonine
Mixture of both species
OPO3H2
OH
NH
H
NH
NH
O
NH
NH
O
R
+ H3PO4
NH
O
R
-101
- 83
-181
yx
y*x+1
yx
yx+1
yx+1
83
R
101
181
m/z
MS Detection of Phosphopeptides
Several ways by ESI:
- Neutral loss scan:
(–98) with triple quad or linear trap. Once the
precursor ion is identified can perform MS/MS and identify peptide
and site of phosphorylation.
-Precursor ion scan: monitor loss of phosphate ion in negative
ion mode
- FT ICR and ECD:
Cleavages at amide bonds leaving phospophates attached
to amino acids.
Easier to determine sites of phosphorylation
Phospho-Specific Hydrolysis of Phosphopeptides
Converts phophopeptides to aminoethylcysteines
by b-elimination (to dehydro-Ala) followed by addition
of aminoethylthiol: get both R and S isomer
Treat derivatized proteins with Lys-C or trypsin:
get cleavage at Lys sites and at phosphorylation sites
(now present at aminoethylcysteine)
Analysis by MS and MS/MS clearly identifies phosphorylation
sites. Only the S isomer is cleaved by the enzyme
Nature Biotech. 2003 21, 1047 - 1054
Phospho-Specific Hydrolysis of Phosphopeptides
NH2
NH2
HSCH2CH2NH2
S
NH
H
O
NH
NH
NH
NH
R
H
O
NH
R
H
O
R
D,L-Lysine
Trypsin
NH2
NH2
S
H
S
OH
NH
NH
NH
O
L (only)
Shokat, Nature Biotech 2003, 21 1047
H
O
D
R
Phospho-Specific Hydrolysis of Phosphopeptides
Nature Biotech. 2003 21, 1047-1054
Quantitation of Phosphoproteins
Control
Experiment
Trypsin H216O
Trypsin H218O
O18
O18
4 Da
O18
IMAC
Phosphatase
O18
O18
~ 40
PNAS 2003 100(3) 880.
~ 60
Time Course Study of Phosphorylation with SILAC
Nature Biotech 2004 22(9) 1139
Time Course Study of Phosphorylation
Nature Biotech 2004 22(9) 1139
PTMs: Glycoproteins
Much more complex:
• O-linked (Ser/Thr), N-Linked (Asn), or both
• Microheterogeneity: same protein may have different
glycoforms
PTMs : Glycoproteins
Can use speficic enzymes to determine nature of sugars
and linkages (a,b) :
a-mannosidase
a-glycosidase,etc..
O-linked: -can undergo b-elimination:
complicates pSer/pThr analysis)
-no specific O-glycanase to cleave from Se/Thr
N-linked: -Can remove entire sugar with PNGase F
(Asn to Asp)
-EndoF cleaves between the first two sugars
Glycoproteins: most common fragmentation
Y1
Y2
Z1
OH
O
A1
O
OH
B1
Non reducing end
C1
OH
O
OH
B2
C2
Z4
O
OH
O
OH
OH
Y4
Z3
HO
O
O
HO
Y3
HO
HO
HO
Z2
O
OH
B3
C3
B4
C4
Reducing end
Use analogous nomenclature to peptides with capital letters
Y, B, C, Z, etc
In certain conditions, can get “cross-ring” fragmentation (A1)
MS/MS of Glycopeptide from RNAse B
MS/MS fragmentation of Glycopeptides
a)163.0
b)457.15
c)660.24
d)1009.36
g)1222.21 h)1154.52
e)990.43
f)997.94
i)1038.15
(Peptide+HexNAc) 2+
j)994.43
(Peptide) 2+
j
e
990.69
994.69
1031.21
c
a
f
998.19
g
1222.54
163.06
i
b
457.15
Pentose (P)
961.68
h
Hexose (H )
1173.87 1282.25
1038.47
HexNAc (N)
Fucose (F)
Pentose (P)
Mannose(M)
Hexose (H)
HexNAc (N)
Fucose (F)
N2H3FX
N2H2NFX
N2H3NFX
N2H3N2FX
N2H3NHF2X
N2H3N2HF2X
N2H3N2H2F3X
N2H3N3H3F2X
N2H3N3H3F4X
Pentose (P)
Deoxyhexose (F)
Hex (H)
HexNAc (N)
132.042
146.057
162.052
203.079
Identification of O-Linked Glycopeptides
S
2 7 4 .1 0
100
A
2 9 2 .1 2
HS
NH
292.1+
NS
4 5 4 .1 8 4 9 5 .2 0
0
100
1142.5++
1471.1++
NHS2
6 5 8 .2 7
9 1 6 .5 1 9 4 8 .3 8 1 0 1 8 .0 5 1 0 9 8 .5 5
8 2 9 .7 4
5 8 3 .2 3
1390.2++
1 2 4 4 .6 7 1 3 2 5 .1 6
m /z
200
300
400
500
600
700
100
B
%
H
T* (peptide)
4 9 6 .2 0
b4
2 9 3 .1 2 3 6 7 .1 6
429.2
2 0 4 .0 9
N
NHS
6 5 7 .2 6
N
454.2+
S
3 6 6 .1 6
%
948.3+
S
x1 0
b7
770.44
771.46
710.44
712.95 733.01
746.27
714.43
1000
1100
2+
Pep+NS
1200
830.08
830.74
871.96
884.12
2+
y12+NS
1300
1400
2+
y12+N2S
960.51
2+
Pep+NHS
915.97
884.46
858.94
997.52
940.00
895.51
914.53
787.06
851.49
1500
1+
2+
NHS2 948.38 Pep+N2H
916.51
872.47 3+
Pep+N2H2S2
829.74
814.43
900
2+
Pep+N2
3+
Pep+N2HS2
2+
b12 +N2
/(y12 +N2)
2+
Pep+N
800
960.98
985.53
998.05
961.53
794.46
0
m/z
2+ 720
Pep+N2S
100
C
1018.05
740
1079.55
760
780
1098.55
800
2+
y12+N2HS
1099.59 2+
Pep+N2S2
2+
Pep+N2H2S
1077.22
860
880
1100.07
1122.66
1072.53
920
2+
Pep+N2HS2
1230.60
960
980
2+
Pep+N2H2S2
2+
y12+N2H2S
1188.08
940
1000
1+
Pep
1244.67
1244.14
1163.61
1186.61
2+
1163.10
1187.58
y12+N2H2S
1042.05
1075.24
900
2+
y12+N2HS2
1078.20
%
840
2+
Pep+N2HS
1018.53
1078.45
820
1268.12
1268.61
1269.21
2+
Pep+N2H2S3
2+
Pep+N2HS3 2+
y12+N2H2S2
1325.16
1326.60
1390.19
1413.70
1389.63
1471.09
0
1025
1050
1075
1100
1125
1150
1175
1200
1225
1250
1275
1300
1325
1350
1375
1400
1425
1450
m/z
1475
FT ICR MS/ECD of Glycoproteins
Quantitation of Glycoproteins
Quantitation
Hiroyuki Kaji et al Nature Biotech 2003,(6) 667-672
1) Trypsin and wash
2) Succinic anhydride
d0 or d4
3) Mix samples
4) PNGase F
Quantitative analysis of succinyl peptides (labeled vs unlabled)
Hui Zhang et al, Nature Biotech 2003 (6) pp 660 - 666
Biological Mass Spectrometry
Many different MS tools now available to perform all sorts
of more and more complex biochemical experiments.
In recent years major improvement in sensitivity, resolution,
mass accuracy, throughput, etc.. However some instruments are
better at certain tasks than others (ie peptides vs intact proteins,
PTMs vs quantitation).
The MS tools as well as software and separation techniques
(eg HPLC-Chips) are improving at a very rapid pace. The combination
of these new tools with classical biochemical approaches are
especially powerful.