CH 908: Mass Spectrometry
Lecture 12
Derivatization of Proteins/Peptides:
Purposes
Separation
Detection/Analysis
 To improve chromatographic
 To improve sensitivity
properties:
- Resolution;
- Retaining
 Analyte isolation/
Sample clean-up
(UV, LIF, MS);
 To improve analyte stability;
 Differential analysis & Quantitation
 To improve MS/MS sequencing
(directed fragmentation)
Multi-function tags – combine 2 or more functions - are preferred!
Labels – widely used and commercially available
The Molecular
Probes Handbook —
A Guide to
Fluorescent Probes
and Labeling
Technologies
Labels
Linkers
(all chemistries for
various target molecules
or objects)
Classification of current
quantitative proteomic techniques
Spiking with an isotopically
labeled analog
M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136
Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company
Differential Gel Electrophoresis
(In-gel Quantitation)
Electrophoresis. 1997 Oct; 18(11): 2071-7.
Unlü M, Morgan ME, Minden JS.
Difference gel electrophoresis: a single
gel method for detecting changes in
protein extracts.
Isotope dilution mass spectrometry
(IDMS)
 IDMS is the use of an enriched isotope of the element of interest
as the internal standard;
 Has been known for nearly 50 years;
 The IDMS technique involves the addition of a known amount of
an enriched isotope of the element of interest to the sample.
- made prior to sample preparation
- the sample concentration can be calculated by measuring the
isotope ratio of the sample and sample + spike;
 Not applicable to monoisotopic elements
 First work cited for peptides: Desiderio DM, Kai M. Preparation
of stable isotope-incorporated peptide internal standards for
field desorption mass spectrometry quantification of peptides in
biologic tissue. Biomed Mass Spectrom. 1983 Aug;10(8):471-9;
Labeling with stable isotopes:
MS and LC views
Mass spectrometry–based proteomics turns quantitative
Shao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005)
Labeling with stable isotopes
 High labeling efficiency
(ideally 100%) is a key
for successful
quantitation
 The earlier the label is introduced – the lesser
uncompensated quantitation errors
 13C, 15N, 18O are preferred to D (less isotopic separation)
Isotopic separation (H/D)
(A) R = 0, ratioobs does not vary with time
and always equals ratiotrue.
(B) R = 0.025, the deuterated peptide (·)
would elute 1.5 s faster, and ratioobs
varies continuously across the peak.
(C) R = 0.5, the deuterated peptide (·)
would elute 30 s faster than the
nondeuterated peptide (▪), and
ratioobs varies continuously across the
peak and is very high
H/D-labeled pairs may separate as
much as 1 min in RP HPLC
=> 13C, 15N, 18O are preferred to D
Fractionation of Isotopically Labeled Peptides in Quantitative
Proteomics. R. Zhang, C. S. Sioma, S. Wang, and F. E. Regnier
Anal. Chem., 2001, 73 (21), pp 5142–5149
Types of labeling workflows in
quantitative proteomics
Least prone
to errors
Uncompensated
quantitation
errors
Mass spectrometry–based proteomics turns quantitative
Shao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005)
Spiking with an
internally labeled stable
isotope standard: The
AQUA strategy
Absolute quantification of proteins
and phosphoproteins from cell
lysates by tandem MS
Scott A. Gerber, John Rush, Olaf
Stemman, Marc W. Kirschner, and
Steven P. Gygi
PNAS 2003;100:6940-6945
Classification of current
quantitative proteomic techniques
Spiking with an isotopically
labeled analog
M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136
Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company
In-vivo metabolic labeling (SILAC)
 SILAC stands for Stable Isotope LAbeling in Culture
 Stable isotopes, such as 15N or 13C, can be incorporated
into proteins during normal biosynthesis by growing
cells in media supplemented with stable-isotope
containing nutrients (typically AA’s)
 Isotopically labeled amino acids used: Leu D3 (allows
differentiation between Leu and Ile), Met D3, Gly D2,
Arg 13C6, Arg 13C615N4, etc.
A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)
Shao-En Ong and Matthias Mann Nature Protocols , 2650 - 2660 (2007)
SILAC scheme
Mumby and Brekken Genome Biology 2005 6:230
SILAC: + and –
Advantages
Limitations
 Up to 100% labeling efficiency
 The main limitation: SILAC is
can be achieved
 The label is introduced at the
earliest possible stage of the
analysis => minimal errors
 Additional sequence
information from ∆m: # of
labeled amino acids can aid in
identification
 The only isotopic labeling
method for top-down protein
quantitation
applicable only for cells which
can be cultivated;
 Mass window between the
two isotopic versions of
peptides is not uniform, and
the data processing tools for
this are not readily available
Top-Down Quantitation and
Characterization of SILAC-Labeled Proteins
 A combination of top-down approach with
quantitation
 Whole proteins can be labeled with efficiency close to
100% (impossible to achieve by chemical labeling due
to hindered access to AA’s in internal regions);
 Complete labeling is a precondition for successful
quantitative top-down analysis
J Am Soc Mass Spectrom. 2007 Nov;18(11):2058-64. Top-down quantitation and characterization of SILAC-labeled proteins.
Waanders LF, Hanke S, Mann M.
Top-Down Quantitation and
Characterization of SILAC-Labeled Proteins
(1)
(2)
(3)
The effects of incomplete isotope enrichment and incomplete mass labeling are modeled, based on the
34+ peak of a theoretical protein of 55 kDa with average amino acid composition, labeled with heavy
Arg and measured with 60,000 resolving power.
(1) A 98% isotope enrichment of 13-C and 15-N in the heavy amino acids results in a shift of the total
heavy isotopic cluster from 100% enrichment. The width and the intensity of the isotopic cluster remain
unchanged.
(2) In contrast, if only 98% of the arginines and lysines are labeled with heavy amino acids, the result is
a significant spread of the signal.
(3) With 95% labeling the signal is even more spread and the total intensity is reduced to 33% of the
original signal.
Classification of current
quantitative proteomic techniques
Spiking with an isotopically
labeled analog
M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136
Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company
Enzymatic
18
( O)
labeling
 The proteolytic enzymes catalyze the following two reactions:
 Proteases can catalyze incorporation of either 1 or 2 18O atoms
Enzymatic labeling example
Partial spectrum of the labeled and
unlabeled dimer of HSP peptide
CLNRQLpSSGVSE. Inset shows
theoretical distribution of
molecular ion envelope for the
unlabeled dimer.
Proteolytic 18O Labeling for Comparative Proteomics: Evaluation of Endoprotease Glu-C as the Catalytic
Agent K. J. Reynolds, X. Yao, and C. Fenselau, Journal of Proteome Research, 2002, 1 (1), pp 27–33
Enzymatic labeling: + and –
Advantages
 Applicable to all types
Limitations
 Small ∆m (2 or 4) => isotopic envelope
overlap;
of biological samples;
nd 18O
 Effective with very low  Incomplete incorporation of 2
sample amounts (as low atom, the reaction is hard to control, and
the rate of exchange differs with peptide
as 1-4 μg of total
size, type of amino acid, between
protein, or 10,000 cells,
enzymes and with peptide sequence;
in a recent report)
 C-terminal peptides are not labeled =>
 No side reactions and
singlets in MS => can be interpreted as
byproducts, which are a
having arisen from large changes in
expression;
general problem of
chemical labeling.
 Losses during sample prep (dry out H2O,
then reconstitute in 18H2O...
Classification of current
quantitative proteomic techniques
Spiking with an isotopically
labeled analog
M.Miyagi , K.C. S. Rao Mass Spectrom. Rev. Vol.26, 1 Pages: 121-136
Copyright © 2007 Wiley Periodicals, Inc., A Wiley Company
Chemical labeling
 Label is an isotope-bearing molecule introduced by a
chemical reaction
 Multifunctional labels can be designed
Julka, S.; Regnier, F. J. of Proteome Res. 2004, 3, 350-363
Martin Münchbach,
Manfredo Quadroni,
Giovanni Miotto, and
Peter James
Quantitation and
Facilitated de Novo
Sequencing of
Proteins by Isotopic
N-Terminal
Labeling of
Peptides with a
FragmentationDirecting Moiety
Anal. Chem., 72 (17),
4047 -4057, 2000
ICAT (Isotope Coated Affinity Tags)
st
1 generation
Reacts with
SH-groups of
Cys
For affinity isolation of
labeled peptides using an
avidin LC column
Isotope label carrier
Gygi, S. P., B. Rist, et al. (1999). "Quantitative analysis of complex protein mixtures using
isotope-coded affinity tags." Nat Biotechnol 17(10): 994-9.
ICAT analysis flowchart
Gygi, S. P.; Aebersold, R. Curr. Opin. Chem. Biol. 2000, 4, 489-94.
ICAT: + and –
Advantages
Limitations
 Multipurpose!
 H/D labeled peptides did not coelute
 Labeling protocol – alkylation (well-
developed);
 Affinity isolation: simplified peptide
during RP HPLC => less accurate
quantitation;
 Bulky biotin molecule (m~500) shifted the
masses of larger peptides outside the
mixtures; presence and # of Cys aids
optimal mass range and also impaired the
identification (constraint)
MS/MS CID spectra of the peptides;
 The only commercially available
method selective for a specific residue
(Cys); enrichment allows proteomic
studies on a much wider dynamic
range than the other methods
 For peptides containing 2 Cys, ∆m (2 x 8=
16) overlapped with the same oxidized
peptide (∆M = 16);
 Not suitable for proteins which do not
contain Cys (e.g., ~8% of the yeast
proteome)
New generation of ICAT : a
cleavable reagent
Applied Biosystems
Cleavable ICAT reagent
Advantages compared to the old ICAT reagent

13C
instead of D => co-elution of ICAT labeled pairs from RP HPLC =>
improved quantification;
 Acid-cleavable site in the reagent => removal of the biotin portion of the ICAT
reagent tag prior to MS and MS/MS analysis;
 Reduced tag fragmentation => improved quality of MS/MS data;
 ∆m of 9 Daltons avoids possible confusion between oxidized methionine and
two cysteines labeled with ICAT reagents
 The number of. proteins detected with this second-generation cleavable
reagent was larger than with the first generation reagent
Investigation of the Linearity, Recovery and
Precision of LC/Triple-Quad MRM-MS/MS with Six
cICAT-Peptides Derived from BSA
A The
linearity for BSA quantification was calculated independently for the six cICAT-peptides over the range of 12–1200
fmol BSA on column (assuming 100% efficiency through cICAT procedures).
B The recovery and precision were measured with the same fetal bovine serum sample (0.1μg total proteins); the recoveries
were determined in triplicate, by spiking respectively at the level of 48 and 480 fmol BSA (on column) into samples; to
determine precision, aliquots of the serum sample stored at −80°C were injected twice on two different days (n=4).
C Detection limits of both systems were defined as the BSA amount on column (assuming 100% efficiency through cICAT
procedures) that gave a S/N of 3; the conditions for quantitative LC/MS/MS were optimized using cICAT-peptides derived
from 50μg/mL BSA.
Total amount of protein used in various cICAT studies ranged from 4.4 mg to 200 µg.
Utility of Cleavable Isotope-Coded Affinity-Tagged Reagents for Quantification of Low-Copy Proteins Induced by
Methylprednisolone Using LC/ MS/MS J. Qu, W. J. Jusko, R. M. Straubinger Anal. Chem., 2006, 78 (13), pp 4543–4552
Targeting phosphopeptides: PhIAT
Phosphoprotein Isotope-Coded Affinity Tag Approach for Isolating and Quantitating Phosphopeptides in
Proteome-Wide Analyses Michael B. Goshe, Thomas P. Conrads, Ellen A. Panisko, Nicolas H. Angell, Timothy D.
Veenstra, and Richard D. Smith Anal. Chem. 73 (11), 2578 -2586, 2001
Tags for MS/MS analysis: iTRAQ
 A complimentary method to ICAT rather than an alternative to it;
 Targets N-termini of peptides as an N-hydroxysuccinimide (NHS-)
ester;
 4 isotopic variants, yet the tag is ISOBARIC in the MS mode; the
difference shows up only after fragmentation of the tag in MS/MS
mode
- 4 samples can be run simultaneously;
- single precursor selection.
Isobaric tags (iTRAQ)
Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents
Ross PL, ... Pappin D.J.(2004) Mol Cell Proteomics 3(12): 1154-69
iTRAQ (continued)
iTRAQ for quantitation
Reporter ion region in a MS/MS spectrum
A tag that influences MS signal and MS/MS
fragmentation
O
 N-methylpiperazine (and similar structures, such as morpholine) is a weak base
 => additional protonation site
 => N-terminal labeling with basic tags leads to enhancement of peptide signals in
the MS mode and also facilitates peptide fragmentation
 => More abundant b-ion series and decreased number of less informative internal
cleavage fragments
 => More sequence ladder fragments and improved MS/MS spectra assignment
 => Higher confidence in protein ID
Conversion of lysine to homoarginine
 Derivatizing agent - O-methylisourea;
 Increase signal intensities of Lys-
containing peptides in MALDI–TOF MS;
 Conversion efficiency for ε-amino groups
of Lys is 100%, side reactions are
minimal, and the reagents are
inexpensive and readily available
 Commercialized as Lys-Tag 4H (Agilent)
Mass defect labeling
 Mass Difference from Nucleon Value of the Most
Abundant Isotope of the Elements Found in Proteins
Element
mass defect (amu)
12C
0
1H
0.0078
16O
−0.0051
15N
0.0031
32S
−0.0279
Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses. H. Hernandez,
S.Niehauser, S.A. Boltz, V. Gawandi, R. S. Phillips, and I. J. Amster, Anal Chem. 2006 May 15; 78(10): 3417–3423
Typical MM distribution for tryptic peptides
Histogram of the molecular mass distribution of the predicted tryptic peptides of M.
maripaludis over the range 1500–1503 Da, Illustrating the distribution of mass defects
of peptides. The bin size is 0.01 amu. Peptide masses are observed to cluster in
approximately one-third of the available mass space.
Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses. H. Hernandez,
S.Niehauser, S.A. Boltz, V. Gawandi, R. S. Phillips, and I. J. Amster, Anal Chem. 2006 May 15; 78(10): 3417–3423
Cys-alkylation reaction
(to introduce mass defect label)
MDL (Mass Deficit Label) reagent 2,4-dibromo-(2′-iodo)acetanilide
Mass Defect Labeling of Cysteine for Improving Peptide Assignment in Shotgun Proteomic Analyses. H. Hernandez,
S.Niehauser, S.A. Boltz, V. Gawandi, R. S. Phillips, and I. J. Amster, Anal Chem. 2006 May 15; 78(10): 3417–3423
MS of peptides labeled with MDL
The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis.
Proteins were identified by their accurate mass values and from their nitrogen
stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the
unlabeled peptides.
[the same reference]
Additional constraint: Phosphopeptides
Distinctively large mass defect of phosphorus relative to H, C, and O (~0.3 Da)
has the net result of off-setting the average mass of phosphopeptides to slightly
lower mass than unmodified peptides of the same nominal molecular weight,
often marking a peptide as phosphorylated simply on the basis of its mass.
Stoichiometry of protein complexes
• In a coprecipitation experiment, the goal is
to identify proteins that bind differentially to
wild-type and mutant baits
(bait = studied protein + affinity tag).
• Proteins that bind specifically to the bait or
secondary interactors will give a ratio
indicative of increased binding and
enrichment.
• Proteins that bind unspecifically to the
affinity support or beads will show ratios
similar to the mixing ratio.
• Repeating the experiment with switched
labels should result in inverse ratios, further
increasing the specificity of the assay.
Mass spectrometry–based proteomics turns quantitative
Shao-En Ong & Matthias Mann Nature Chemical Biology , 252 - 262 (2005)
Classification of current
quantitative proteomic techniques
Spiking with an isotopically
labeled analog
We are still here!
MS/MS tags: directed fragmentation
 Why do we need that?
- De novo (old times);
- Improve quality of the MS/MS spectra
 Less than 20% of all peaks in MALDI-TOF MS spectra
and < 5% of ESI-IT yield interpretable MS/MS spectra
which result in peptide identification!
 MS/MS data acquisition takes ~80% of MS work time
MS/MS: The Good…
An Example of a Good Spectrum
Sequence: FGQGEAAPVVAPAPAPAPEVQTK
MASCOT score 252
MS/MS: The Bad…
“OK” Spectrum
QNNFNAVR
Mascot Score 43
MS/MS: The Ugly
Poor fragmentation
Sequence: GALSAVVADSR,
MASCOT score 10
Charge-directed fragmentation
 Fixed-charge N-
terminal tags:
quaternary ammonium
or phosphonium
cations
 First offered for sector
tandem MS
instruments (Biemann
et.al.) in early 1990ies
Charge-directed fragmentation of α-thymosin
α-thymosin
Zaia, J.; Biemann, K., Comparison of charged derivatives for high energy collision-induced dissociation
tandem mass spectrometry Anal. Chem. 1995, 6, 428-436
Charge-directed fragmentation (more spectra)
Zaia, J.; Biemann, K., Comparison of charged derivatives for high energy collision-induced dissociation
tandem mass spectrometry Anal. Chem. 1995, 6, 428-436
Cationic tag labeling for MALDI-TOF/TOF
(“high-energy” collisions)
A
6.2E+4
% Intensity
y9(+1)
100
EGVNDNEEGFFSAR
90
80
Native
70
60
y14(+1)
50
y6(+1)
40
y1(+1)
30
y7(+1)
y9-17(+1)
20 R
y2 - 17(+1)
y10(+1)
10
y2(+1)y3(+1) y4(+1) y5(+1)b7-18(+1) y8(+1)
y11(+1) y13(+1)b14(+1)
0
387.4
704.8 Mass (m/z)
1022.2
1339.6
1657.0
70.0
B
3.7E+4
b*(+1)
EGVNDNEEGFFSAR
TMPP tag
% Intensity
100
90
80
70
60
50
40
30
20
10 77.2654
0
70.0
559.2617
a1(+1)
527.2357 b1(+1)
508.2
No sequence fragments!
1986.8705
b5(+1)
a7(+1)b8(+1)a10(+1)a11(+1)b13(+1)
b4(+1) b6(+1)
946.4
1384.6
Mass (m/z)
1822.8
2261.0
Coumarin Tags for Analysis of Peptides by MALDI-TOF MS and MS/MS. 2. Alexa Fluor 350 Tag for Increased Peptide and Protein
Identification by LC-MALDI-TOF/TOF MS A.Pashkova, (...) E. Moskovets, and B. L. Karger Anal. Chem., 2005, 77 (7), pp 2085–2096
A Mobile Proton Theory of Peptide Fragmentation
 The most stable protonated form may not be the
fragmenting structure
 Fragmentation (backbone) occurs due to the weakening of
the amide bond, i.e. decrease of the bond order
 Calculations showed that this will happen in the case of the
protonation of the amide N
 The more “mobile” (not localised) the proton, the more
fragments in a MS/MS spectrum =>the more information
from the spectrum
Dongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V. H., J. Am. Chem. Soc. 1996, 118, 8365-8374.
Localization of a Proton on a
Protonated Peptide
+
H
Order of basicity:
Guanidine group of Arg > N-term N > Carbonyl O > Amide N
NH2
HN
=> a dynamic equilibrium of structures
+
+
H
H
R1
O
+
H
O
H
N
H2N
NH
R2
N
H
OH
O
The Fragmenting Structure of a Protonated Peptide
The most stable protonated form is NOT the fragmenting
structure!
NH2
HN
+
NH
H
R1
O
H
N
H2N
O
R2
N
H
OH
O
Sulfonated peptide: adding 1 extra
mobile proton
+
NH2
H2N
+
NH
H
O
O
S
O
R1
O
O
H
N
N
O
R2
N
H
OH
O
Tags containing a sulfo-group
H
H N
O
O
Alexa Fluor 350 succinimide ester
O
Added mass 295.01
O Su
HO3S
(Molecular Probes)
Me
O
HO3S
3-sulfopropionic acid succinimide ester
(CAF reagent)
O Su
Added mass 135.62
Keough, T.; Youngquist, R. S.; Lacey, M. P., Anal. Chem. 2003, 75, 156A-165A
Labeling with Alexa Fluor 350
Sequence: GALSAVVADSR,
MASCOT score 10
Monoisotopic mass of neutral
peptide (Mr): 1044.556
Ions Score: 10
Matches (Bold Red):
5/63 fragment ions using 7 most intense
peaks
Mascot Score 95
Monoisotopic mass of neutral
peptide (Mr): 1339.571
Fixed modifications: Guanidination (K)
Variable modifications: N-term : Alexa (N-term)
Ions Score: 95
Matches (Bold Red): 10/63 fragment ions using
11 most intense peaks
Low MS signal intensity precursor
SVDEAANSDIVDK
1570.6772
MS/MS: Mascot score 119
100
90
2.4E+4
80
% Intensity
70
60
50
40
MS: S/N 9, 350 counts
1588.2
1630.4
1672.6
1719.5154
1699.6768
1669.6000
1627.6600
0
1546.0
1604.7000
10
1565.7336
20
1552.6720
30
1714.8
1757.0
Mass (m/z)
High quality spectrum obtained from a MS precursor with S/N< 10
(E.coli protein digest labeled with Alexa Fluor 350)
Complimentary information?
Peptides
Native
295 unique
(34.6%)
Proteins
Alexa Tagged
124
common
(14.5%)
Hydrophobic, Argpeptides
433 unique
(50.8%)
Native
85 unique
(25.2%)
Alexa Tagged
147
common
(43.6%)
105 unique
(31.2%)
Hydrophilic, Lyspeptides
LC-MALDI-TOF/TOF MS analysis of tryptic peptides from the SCX fractions of an E.
coli lysate revealed improved peptide scores, a doubling of the total number of
peptides, and a 30% increase in the number of proteins identified, as a result of
labeling with Alexa Fluor 350.