ionspec. ft icr isotopic resolution of proteins: myoglobin 12 t qft icr ms (uwo)
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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.
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)
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 morecharged to yield singly charged radical ion. Dissociation or fragmentation will occur a the amide bond between the NH and CH (N-C)giving primarily c and z ions. It is therefore complementary to SORIIRMPD and classical CAD in quad collision cell. Another advantage is for the analysis of PTMs since the amide bond is cleaved first not thephospho bond or glycosidic linkage.
H2N C C N
O
R1
HHC C N
O
R2
HHC C N
O
R3
HHC C N
O
R4
HHC C OH
O
R5
H
a1 b1 a2 a3 a4 a5 b2
b4 b3 c2 c3 c4c1
x1 y1 z1x4 x3 x2y4 y3 y2z3z4 z2
H2N C C N
R1
OH HHC C N
O
R2
HH
. H2N C C N
R1
OH HH HHC C N
O
R2
.
z
+
c
H2N C C N
R1
OHHH
C C N
O
R2
HH+
+ e-
81.972+
897.512+
998.552+
712.434+
948.032+
1104.612+
868.512+
1657.78493.31 1832.431187.66 1527.081054.58
2+1272.63
594.37 642.46541.673+
1927.20
0
20
40
60
80
100
400 600 800 1000 1200 1400 1600 1800 2000
712.184+
906.473+
1216.972+
949.113+
969.15
1415.282+
1721.44
1159.992+
1104.022+
656.23
528.211075.06
2+1127.03
863.833+
811.662+
1033.99 1656.25
1336.88 1830.70755.172+
838.641925.301358.84
2+
989.072+
414.15 612.24
1525.88
m/z
Electron capture dissociation (ECD) → c, z ions
Infrared multiphoton dissociation (IRMPD) → b, y ions
Complementary Sequencing from Two Different MS/MS TechniquesJLC
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 thepost-translational modifications which are often missed by bottom-up approaches since coverage tends to be poor 5-30%.
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.
However:Size limitation ~45kDaLimit of detection: larger proteins are more difficult to ionize
Orbitrap
Latest on the market with a new type of mass analyzer: Hybrid instrument combining with a linear trap with the orbitrap
FT MS with no magnet!
FT MSImage current detectorHigh resolutionAccurate mass
2 Detectors in one instrument
FT MS with no magnet!
Orbitrap
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
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 theLinear trap (faster but lower resolution) or in the Orbitrap.
Important new tool in “bottom-up” proteomics
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
-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
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
2. Intensity of signal (normalized)
Quantitative Proteomic Profiling
Stable Isotope labeling2D GE Intensity-based quantitation
In vivo labeling In vitro labeling
N-terminalpeptide labeling
C-terminalpeptide lableling
Amino Acid-Based labeling
N14/N15 media SILAC
Cys:ICATiTRAQ
Esterification(CD3OH)
16O/18O incorporationvia proteolysis
To quantitate expression of proteins from cells exposed to differentconditions 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 !!
ICAT: Isotope Encoded Affinity Tags
Original ICAT Reagent
HN NH
O
X
SNH
O
OX
XX
OO N
X
XX
X
O
I
H
Gygi et al, Nature Biotech (1999) 17 994Aebersold et al, Curr. Opin. Biotech (2003) 14, 110
Light ICAT d0 , X = H
Heavy ICAT d8, X = D
HS-Cys-peptide
Biotin Affinity tag Linker with labels Reactive group
ICAT Procedure
1. Denature (urea +SDS) and reduce proteins (TCEP) (Tris- caboxyethylphophine) HCl2. 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
HS SHSH
SHSH HS
SH
HS
HS SHSH
SHSH HS
SH
HS
SSSSSSS
S
SSS
S S S
SSSSSSS
S
SSS
S S S
Denature and reduce
Labeling with light ICAT Labeling with heavy ICAT
Mix and Trypsin digestion
S
SS
S
S
S
S
SS
S
S
S
Affinity capture biotin/avidin
LC MS MS/MS RatioLight /heavy ID
Non retained
Peptides
ICAT (cont..)
m/z m/z
LC MS
S
S
8 Da
MS/MS
Database Search
(MASCOT)
Identification of protein
Ratio ~ 60:40
L
H
560 568
(281.5)
Select doubly charged ion
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).
ICAT Isotope encoded affinity tags
Isotope tag X = H or D (m = 7)
Zhou et al., Nature Biotech. 2002, 19, 512
Solid Phase ICAT
Glass beadsPhotocleavable linker
Thiol reactive
CXX3C CX3
H
H
H
h cleavageHS-Cys-peptide
N
O
O
CH3 N
O
N
O
I
OCH3
NO2
Better, but still problems: reagent is very light sensitive (and expensive)
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 samplesPerformed 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
Mass difference of 2 (or 4): need high resolution MS !
Get 1 (or 2 O18)
m/z
Inte
nsity
COH
O
Arg (Lys)
18
18
COH
O
Arg (Lys)
18
COH
O
Arg (Lys)
+2 +4
MS of tryptic peptides digest labeled with O18 vs control
100%
Ratio labeled vs unlabeled ~2:1
With Arg can get exclusively + 4 DaWith Lys get a mixture of + 2 and + 4 Da
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
Quantitation: Labeled Amino Acids
Limitations: Some ScramblingNot generally applicable to all types of experimtns (eg Human tissues)
J. Proteome Research 2002,1 , 345-350Rapid Comm. Mass Spec. 2002,16 2115-2123
SILAC (stable isotope labeling with amino acidsIn cell culture)
Leu-d3
SILAC Procedure
Ong, S.-E. (2002) Mol. Cell. Proteomics 1: 376-386
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
Labeling with 15N or 13C
Gives complex mixturedifficult to interpret
Each peptide is enrichedwith 15N and/or 13C
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.
NH -peptide
Reporter group114,115,116.117
Balance group31,30,29,28
MS/MS Fragmentation site
Isobaric tag mass = 142
N N
N
CH3
CH3
O
Y Y= leaving group
N N
N
CDH2
H2DC
O
Y
N N
N
CH3
H2DC
O
Y
N N
N
CD2H
H2DCO
Y
*
*
*
*
114 115
116117
31 30
2928
The Four iTRAQ Reagents
N N
N
O
HN
R
O
OH*
*
N N
N
C
O
HN
R
O
OH
*
*+
CID
PeptideFragments
Quantifying Fragment
m/z 114 32 Da Peptide Identifciation
iTRAQ: Procedure
Up to 4 test samples
ITRAQ: Advantages and Disadvantages
-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.
Advantages
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 +80 Enzyme Activity, signalingpTyr, +++pSer, pThr +/++Glycosylation Cell/cell recognition N-linked >800 ++ O-Linked >203 ++Acetylation +42 +++ Protein stabilityMethylation +14 +++ Gene expressionAcylation Cellular Localization Farnesyl +204 +++ Myristoyl +210 +++
Sulfation +80 Protein/protein interactionsDisulfide -2 ++ Protein stabilityDeamidation +1 +++ Protein/protein interactions, agingUbiquitination >1000 +++ Protein degradationNitration +45 ++ Oxidative damageTruncation -x +++
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-stoichiometrypoor ionization and ion suppressionother 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
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
Detection of Phosphopeptides at Low Levels
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
D. Kalume et al Curr. Opinion in Chem. Biol. 2003, 7:64-69
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 -elimination to dehydro-Ala (-98) which can react with nucleophile (thiols, amines) enrichment by IMAC
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
N
H2C
H2C
C
C
O
O
O
O
M CH
C
CH2
O
OP
OH
O
O
NH
CH
C
CH3
OH
O
HN
CH
C
H2C
HOO
OP
OH
O
O
IMAC
M = Fe , Ga
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)
NH
OPO3H2
O
NHH
R
:Base
NH
O
NH
R
+ H3PO4
-Elimination
-98
NH
OH
O
NHH
R
O
Ba(OH)2
-69
MS/MS
-87
Dehydroalanine
Serine
Phosphoserine and -elimination
Phosphate group of serine is very labile and can be -eliminated by base treatment OR in the MS experiment
NH
O
NH
R
+ H3PO4
69
yx y*x+1
NH
OH
O
NHH
R
O
87
yx yx+1
MS/MS of Phosphoserine vs Serine
m/zm/z
dehydroalanineserine
NH
OPO3H2
O
NHH
R
MS/MS of PhosphoThreonine vs Threonine
83
yx y*x+1
101
yx yx+1
m/z
yx+1
181
Mixture of both species
-181
NH
O
NH
R
OH
NH
O
NH
R+ H3PO4
- 83 -101
MS Detection of Phosphopeptides
Several ways by ESI:
- Neutral loss scan: (–98) with triple quad or linear trap. Once theprecursor ion is identified can perform MS/MS and identify peptide and site of phosphorylation.
- FT ICR and ECD:
-Precursor ion scan: monitor loss of phosphate ion in negative ion mode
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 -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
NH
O
NH
R
HSCH2CH2NH2
H
NH
O
NH
RH
S
NH2
NH
O
NH
RH
NH2
D,L-Lysine
NH
O
S
OHH
NH2
NH
O
NH
R
S
NH2
D
H
L (only)
Trypsin
Phospho-Specific Hydrolysis of Phosphopeptides
Shokat, Nature Biotech 2003, 21 1047
Nature Biotech. 2003 21, 1047-1054
Phospho-Specific Hydrolysis of Phosphopeptides
Trypsin H216O Trypsin H2
18O
O18
O18
O18
IMAC
Phosphatase
O18
O18
4 Da
PNAS 2003 100(3) 880.
Quantitation of Phosphoproteins
Control Experiment
~ 60~ 40
Time Course Study of Phosphorylation with SILAC
Nature Biotech 2004 22(9) 1139
Nature Biotech 2004 22(9) 1139
Time Course Study of Phosphorylation
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) :
-mannosidase-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
HO
O
OOH
OH
HO
O
OOH
OH
HO
O
OOH
OH
HO
O
OOH
OH
HO
Non reducing end
A1 B1 B3B2B4
Y1 Y4Y3Y2
C1 C2 C3 C4
Z1 Z2 Z3 Z4
Reducing end
Glycoproteins: most common fragmentation
Use analogous nomenclature to peptides with capital lettersY, B, C, Z, etc
In certain conditions, can get “cross-ring” fragmentation (A1)
MS/MS of Glycopeptide from RNAse B
994.69
a)163.0 b)457.15 c)660.24 d)1009.36
e)990.43 f)997.94
g)1222.21 h)1154.52 i)1038.15
j)994.43
1031.21961.68
990.69
1173.87163.06 1282.25
1222.54a
c
j
h
457.15b 1038.47
i
e
998.19f
g
994.69
a)163.0 b)457.15 c)660.24 d)1009.36
e)990.43 f)997.94
g)1222.21 h)1154.52 i)1038.15
j)994.43
a)163.0 b)457.15 c)660.24 d)1009.36
e)990.43 f)997.94
g)1222.21 h)1154.52 i)1038.15
j)994.43
1031.21961.68
990.69
1173.87163.06 1282.25
1222.54a
c
j
h
457.15b 1038.47
i
e
998.19f
g
MS/MS fragmentation of Glycopeptides
(Peptide) 2+
(Peptide+HexNAc) 2+
Pentose (P) Hexose (H ) HexNAc (N) Fucose (F)
N2H3FX
N2H2NFX
N2H3NFX
N2H3N2FX
N2H3NHF2X
N2H3N2HF2X
N2H3N2H2F3X
N2H3N3H3F2X
N2H3N3H3F4X
Pentose (P) Mannose(M) Hexose (H) HexNAc (N) Fucose (F)
Pentose (P) 132.042Deoxyhexose (F) 146.057
Hex (H) 162.052HexNAc (N) 203.079
1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0
1 0 0
%
x 1 02 7 4 . 1 0
2 0 4 . 0 9
2 9 2 . 1 2
4 9 5 . 2 04 5 4 . 1 8
3 6 6 . 1 6
2 9 3 . 1 2 3 6 7 . 1 6
6 5 7 . 2 6
4 9 6 . 2 0
5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7
9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6
b 44 2 9 . 2
N
S
N H
H S N S
N H S
N H S 2
N
HS
S
T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +
9 4 8 . 3 +
1 1 4 2 . 5 + +
1 3 9 0 . 2 + +1 4 7 1 . 1 + +
7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0
1 0 0
%
9 4 8 . 3 8
9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4
7 7 0 . 4 47 1 0 . 4 4
7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7
8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6
7 9 4 . 4 6
8 3 0 . 0 8 8 7 1 . 9 6
8 5 8 . 9 48 3 0 . 7 4
8 5 1 . 4 9
8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6
8 9 5 . 5 19 1 4 . 5 3
9 4 0 . 0 0
9 6 0 . 5 1
9 9 7 . 5 2
9 8 5 . 5 39 6 0 . 9 8
9 6 1 . 5 39 9 8 . 0 5
b 7
2 +P e p + N
2 +b 1 2 + N 2/ ( y 1 2 + N 2 )
3 +P e p + N 2 H S 2
2 +y 1 2 + N S
2 +P e p + N 2
3 +P e p + N 2 H 2 S 2
2 +P e p + N S
1 +N H S 2
2 +y 1 2 + N 2 S
2 +P e p + N 2 H
2 +P e p + N H S
1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0
1 0 0
%
1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5
1 0 1 8 . 5 31 0 7 8 . 4 5
1 0 7 8 . 2 0
1 0 7 7 . 2 2
1 0 4 2 . 0 51 0 7 5 . 2 4
1 0 7 2 . 5 3
1 0 9 9 . 5 91 2 4 4 . 6 7
1 2 4 4 . 1 4
1 1 6 3 . 6 11 1 0 0 . 0 7
1 1 6 3 . 1 0
1 1 2 2 . 6 6
1 1 8 6 . 6 11 1 8 7 . 5 8
1 1 8 8 . 0 8
1 2 3 0 . 6 0
1 3 2 5 . 1 6
1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1
1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9
2 +P e p + N 2 S
2 +y 1 2 + N 2 H S
2 +P e p + N 2 H S
2 +y 1 2 + N 2 H 2 S
2 +P e p + N 2 S 2
2 +P e p + N 2 H 2 S
2 +y 1 2 + N 2 H S 2
2 +P e p + N 2 H 2 S 2
2 +y 1 2 + N 2 H 2 S 2 +
y 1 2 + N 2 H 2 S 2
2 +P e p + N 2 H S 2
2 +P e p + N 2 H S 3
2 +P e p + N 2 H 2 S 3
A
C
B
1 +P e p
1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0
1 0 0
%
x 1 02 7 4 . 1 0
2 0 4 . 0 9
2 9 2 . 1 2
4 9 5 . 2 04 5 4 . 1 8
3 6 6 . 1 6
2 9 3 . 1 2 3 6 7 . 1 6
6 5 7 . 2 6
4 9 6 . 2 0
5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7
9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6
b 44 2 9 . 2
N
S
N H
H S N S
N H S
N H S 2
N
HS
S
T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +
9 4 8 . 3 +
1 1 4 2 . 5 + +
1 3 9 0 . 2 + +1 4 7 1 . 1 + +
7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0
1 0 0
%
9 4 8 . 3 8
9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4
7 7 0 . 4 47 1 0 . 4 4
7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7
8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6
7 9 4 . 4 6
8 3 0 . 0 8 8 7 1 . 9 6
8 5 8 . 9 48 3 0 . 7 4
8 5 1 . 4 9
8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6
8 9 5 . 5 19 1 4 . 5 3
9 4 0 . 0 0
9 6 0 . 5 1
9 9 7 . 5 2
9 8 5 . 5 39 6 0 . 9 8
9 6 1 . 5 39 9 8 . 0 5
b 7
2 +P e p + N
2 +b 1 2 + N 2/ ( y 1 2 + N 2 )
3 +P e p + N 2 H S 2
2 +y 1 2 + N S
2 +P e p + N 2
3 +P e p + N 2 H 2 S 2
2 +P e p + N S
1 +N H S 2
2 +y 1 2 + N 2 S
2 +P e p + N 2 H
2 +P e p + N H S
1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0
1 0 0
%
1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5
1 0 1 8 . 5 31 0 7 8 . 4 5
1 0 7 8 . 2 0
1 0 7 7 . 2 2
1 0 4 2 . 0 51 0 7 5 . 2 4
1 0 7 2 . 5 3
1 0 9 9 . 5 91 2 4 4 . 6 7
1 2 4 4 . 1 4
1 1 6 3 . 6 11 1 0 0 . 0 7
1 1 6 3 . 1 0
1 1 2 2 . 6 6
1 1 8 6 . 6 11 1 8 7 . 5 8
1 1 8 8 . 0 8
1 2 3 0 . 6 0
1 3 2 5 . 1 6
1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1
1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9
2 +P e p + N 2 S
2 +y 1 2 + N 2 H S
2 +P e p + N 2 H S
2 +y 1 2 + N 2 H 2 S
2 +P e p + N 2 S 2
2 +P e p + N 2 H 2 S
2 +y 1 2 + N 2 H S 2
2 +P e p + N 2 H 2 S 2
2 +y 1 2 + N 2 H 2 S 2 +
y 1 2 + N 2 H 2 S 2
2 +P e p + N 2 H S 2
2 +P e p + N 2 H S 3
2 +P e p + N 2 H 2 S 3
A
C
B
1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0
1 0 0
%
x 1 02 7 4 . 1 0
2 0 4 . 0 9
2 9 2 . 1 2
4 9 5 . 2 04 5 4 . 1 8
3 6 6 . 1 6
2 9 3 . 1 2 3 6 7 . 1 6
6 5 7 . 2 6
4 9 6 . 2 0
5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7
9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6
b 44 2 9 . 2
N
S
N H
H S N S
N H S
N H S 2
N
HS
S
T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +
9 4 8 . 3 +
1 1 4 2 . 5 + +
1 3 9 0 . 2 + +1 4 7 1 . 1 + +
1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0m / z0
1 0 0
%
x 1 02 7 4 . 1 0
2 0 4 . 0 9
2 9 2 . 1 2
4 9 5 . 2 04 5 4 . 1 8
3 6 6 . 1 6
2 9 3 . 1 2 3 6 7 . 1 6
6 5 7 . 2 6
4 9 6 . 2 0
5 8 3 . 2 31 0 9 8 . 5 51 0 1 8 . 0 56 5 8 . 2 7
9 4 8 . 3 89 1 6 . 5 18 2 9 . 7 4 1 2 4 4 . 6 7 1 3 2 5 . 1 6
b 44 2 9 . 2
N
S
N H
H S N S
N H S
N H S 2
N
HS
S
T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +
9 4 8 . 3 +
1 1 4 2 . 5 + +
1 3 9 0 . 2 + +1 4 7 1 . 1 + +
N
HS
S
T * ( p e p t i d e )N
HS
S
T * ( p e p t i d e )2 9 2 . 1 + 4 5 4 . 2 +
9 4 8 . 3 +
1 1 4 2 . 5 + +
1 3 9 0 . 2 + +1 4 7 1 . 1 + +
7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0
1 0 0
%
9 4 8 . 3 8
9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4
7 7 0 . 4 47 1 0 . 4 4
7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7
8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6
7 9 4 . 4 6
8 3 0 . 0 8 8 7 1 . 9 6
8 5 8 . 9 48 3 0 . 7 4
8 5 1 . 4 9
8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6
8 9 5 . 5 19 1 4 . 5 3
9 4 0 . 0 0
9 6 0 . 5 1
9 9 7 . 5 2
9 8 5 . 5 39 6 0 . 9 8
9 6 1 . 5 39 9 8 . 0 5
b 7
2 +P e p + N
2 +b 1 2 + N 2/ ( y 1 2 + N 2 )
3 +P e p + N 2 H S 2
2 +y 1 2 + N S
2 +P e p + N 2
3 +P e p + N 2 H 2 S 2
2 +P e p + N S
1 +N H S 2
2 +y 1 2 + N 2 S
2 +P e p + N 2 H
2 +P e p + N H S
7 2 0 7 4 0 7 6 0 7 8 0 8 0 0 8 2 0 8 4 0 8 6 0 8 8 0 9 0 0 9 2 0 9 4 0 9 6 0 9 8 0 1 0 0 0m / z0
1 0 0
%
9 4 8 . 3 8
9 1 6 . 5 18 7 2 . 4 78 2 9 . 7 4
7 7 0 . 4 47 1 0 . 4 4
7 1 2 . 9 5 7 3 3 . 0 17 1 4 . 4 3 7 4 6 . 2 7
8 1 4 . 4 37 7 1 . 4 6 7 8 7 . 0 6
7 9 4 . 4 6
8 3 0 . 0 8 8 7 1 . 9 6
8 5 8 . 9 48 3 0 . 7 4
8 5 1 . 4 9
8 8 4 . 1 2 9 1 5 . 9 78 8 4 . 4 6
8 9 5 . 5 19 1 4 . 5 3
9 4 0 . 0 0
9 6 0 . 5 1
9 9 7 . 5 2
9 8 5 . 5 39 6 0 . 9 8
9 6 1 . 5 39 9 8 . 0 5
b 7
2 +P e p + N
2 +b 1 2 + N 2/ ( y 1 2 + N 2 )
3 +P e p + N 2 H S 2
2 +y 1 2 + N S
2 +P e p + N 2
3 +P e p + N 2 H 2 S 2
2 +P e p + N S
1 +N H S 2
2 +y 1 2 + N 2 S
2 +P e p + N 2 H
2 +P e p + N H S
1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0
1 0 0
%
1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5
1 0 1 8 . 5 31 0 7 8 . 4 5
1 0 7 8 . 2 0
1 0 7 7 . 2 2
1 0 4 2 . 0 51 0 7 5 . 2 4
1 0 7 2 . 5 3
1 0 9 9 . 5 91 2 4 4 . 6 7
1 2 4 4 . 1 4
1 1 6 3 . 6 11 1 0 0 . 0 7
1 1 6 3 . 1 0
1 1 2 2 . 6 6
1 1 8 6 . 6 11 1 8 7 . 5 8
1 1 8 8 . 0 8
1 2 3 0 . 6 0
1 3 2 5 . 1 6
1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1
1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9
2 +P e p + N 2 S
2 +y 1 2 + N 2 H S
2 +P e p + N 2 H S
2 +y 1 2 + N 2 H 2 S
2 +P e p + N 2 S 2
2 +P e p + N 2 H 2 S
2 +y 1 2 + N 2 H S 2
2 +P e p + N 2 H 2 S 2
2 +y 1 2 + N 2 H 2 S 2 +
y 1 2 + N 2 H 2 S 2
2 +P e p + N 2 H S 2
2 +P e p + N 2 H S 3
2 +P e p + N 2 H 2 S 3
1 0 2 5 1 0 5 0 1 0 7 5 1 1 0 0 1 1 2 5 1 1 5 0 1 1 7 5 1 2 0 0 1 2 2 5 1 2 5 0 1 2 7 5 1 3 0 0 1 3 2 5 1 3 5 0 1 3 7 5 1 4 0 0 1 4 2 5 1 4 5 0 1 4 7 5m / z0
1 0 0
%
1 0 9 8 . 5 51 0 7 9 . 5 51 0 1 8 . 0 5
1 0 1 8 . 5 31 0 7 8 . 4 5
1 0 7 8 . 2 0
1 0 7 7 . 2 2
1 0 4 2 . 0 51 0 7 5 . 2 4
1 0 7 2 . 5 3
1 0 9 9 . 5 91 2 4 4 . 6 7
1 2 4 4 . 1 4
1 1 6 3 . 6 11 1 0 0 . 0 7
1 1 6 3 . 1 0
1 1 2 2 . 6 6
1 1 8 6 . 6 11 1 8 7 . 5 8
1 1 8 8 . 0 8
1 2 3 0 . 6 0
1 3 2 5 . 1 6
1 2 6 8 . 1 21 2 6 8 . 6 11 2 6 9 . 2 1
1 3 9 0 . 1 91 3 2 6 . 6 01 3 8 9 . 6 3 1 4 1 3 . 7 0 1 4 7 1 . 0 9
2 +P e p + N 2 S
2 +y 1 2 + N 2 H S
2 +P e p + N 2 H S
2 +y 1 2 + N 2 H 2 S
2 +P e p + N 2 S 2
2 +P e p + N 2 H 2 S
2 +y 1 2 + N 2 H S 2
2 +P e p + N 2 H 2 S 2
2 +y 1 2 + N 2 H 2 S 2 +
y 1 2 + N 2 H 2 S 2
2 +P e p + N 2 H S 2
2 +P e p + N 2 H S 3
2 +P e p + N 2 H 2 S 3
A
C
B
1 +P e p
Identification of O-Linked Glycopeptides
FT ICR MS/ECD of Glycoproteins
Quantitation of Glycoproteins
Quantitation
Hiroyuki Kaji et al Nature Biotech 2003,(6) 667-672
Hui Zhang et al, Nature Biotech 2003 (6) pp 660 - 666
1) Trypsin and wash
2) Succinic anhydride
3)
d0 or d4
Mix samples
Quantitative analysis of succinyl peptides (labeled vs unlabled)
4) PNGase F
Biological Mass Spectrometry
Many different MS tools now available to perform all sorts of more and more complex biochemical experiments.
The MS tools as well as software and separation techniques (eg HPLC-Chips) are improving at a very rapid pace. The combinationof these new tools with classical biochemical approaches are especially powerful.
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).
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