18
O labelling of C-termini of cross-linked
peptides using Trypsin and Glu-C
Pascal van Alphen
Swammerdam Institute for Life Sciences (SILS), Mass Spectrometry Group, University of Amsterdam,
Amsterdam, The Netherlands.
The pH dependency of the carboxyl oxygen exchange reaction catalysed by Glu-C has been
studied. This resulted in a protocol for efficiently labelling cross-linked proteins, digested by
more than one protease, by 18O incorporation into the C-termini. Cross links between amino
acid residues in close proximity can provide distance constraints in order to validate computer
models of the 3D structure of proteins. An 18O labelled cross-link differs from unlabelled crosslinks by 8 amu whereas surface-labels (mono-link) or loop-links shift only 4 amu.
Bis(succinimidyl)-3-azidomethyl-glutarate (BAMG) was used to cross-link cytochrome c and
Gas2p, respectively. BAMG is a cross-linking agent with an azido group that allows for
selective and efficient purification of peptide mixtures. Here, it is shown that Glu-C is able to
efficiently label peptides with 18O in conditions similar to those normally chosen for trypsin.
Using this method, several cross-links have been identified in cytochrome c and Gas2p.
Keywords: cross-linking - double digestion - trypsin - Glu-C - 18O labelling - mass spectrometry
Introduction
For the development of drugs, knowledge of
the tertiary or quaternary structure and active site
of a protein or protein complex is of great
importance. The technique most suited for
obtaining knowledge is X-ray diffraction, but
suffers from the difficulty with which proteins
form crystals.
Computer models predicting the structure of
proteins by their amino acid sequence are
becoming more and more sophisticated and may
remove the need for X-ray diffraction. However,
they still require experimental validation.
Chemical cross-linking of surface residues of
proteins can provide distance constraints with
which those models can be validated[1,2].
Unfortunately, flexibility in protein structure is
required for susceptibility to proteolytic
digestion[3] which implies cross-linking can only
be partial and therefore results in a low
abundance of cross-linker-modified peptides.
This hampers efficient analysis by mass
spectrometry and sequence elucidation by
MS/MS.
Recently, a cross-linker was synthesised that
enables selective reactions with modified
peptides in order to purify peptide mixtures[4].
However, this purification does not distinguish
between highly informative cross-links and
surface-labels (modified by cross-linker but not
actually cross-linked).
A problem with cross-linking large proteins
and protein complexes is that the amount of
possible cross-links increases dramatically and
therefore the amount of false positives. An
elegant method to identify actual cross-links has
been described based on the notion that crosslinks have two C-termini that can be labelled
with 18O whereas surface-labels only have one[5].
Labelled cross-links will show a shift of 8 amu
whereas surface-labels will only shift by 4 amu.
It has long been known that trypsin can
catalyse this carboxyl oxygen exchange
1
reaction[6] and evidence has been presented that
Glu-C can catalyse this reaction as well[7].
However, conditions for efficient labelling by
both Glu-C and trypsin have not yet been
defined. A double digestion with Glu-C and
trypsin would be useful as it will increase the
amount of cross-linked peptides in the mass
range optimal for mass spectrometry, i.e. approx.
1000-3000 Da.
In this study, cross-linked horse heart
cytochrome c will be used to define conditions
under which efficient double labelling occurs.
Various BAMG-cross-links in cytochrome c
have been mapped previously[4] which is used for
validation of our method. Subsequently the
technology will be used for Gas2p to identify
new cross-links which will be used to construct a
model for the 3D structure of Gas2p.
Mass spectra will be analysed with
[8]
VIRTUALMSLAB . VIRTUALMSLAB can produce virtual
mass spectra of digested and modified proteins
and is used to find candidate cross-links. The
next step is establishing a generally applicable
protocol for providing distant constraints with
which 3D structures predicted by computer
models can be validated.
Materials and Methods
Materials. Mass spectrometry grade
modified porcine trypsin (Trypsin Gold) was
obtained from Promega (Madison, WI). Glu-C
from S. aureus was obtained from Roche
(Switzerland). Bovine Insulin was purchased
from Sigma. The peptide fragment monitored for
the optimisation of the carboxyl oxygen
exchange reaction catalysed by Glu-C was PheVal-Asn-Gln-His-Leu-Cys-Gly-Ser-His-LeuVal-Glu (1539.7 m/z) from a Glu-C digest of
reduced and alkylated bovine insulin. Insulin
was reduced using tris(2-carboxyethyl)phosphine
(TCEP) and alkylated by iodoacetamide (IAA).
18
O-enriched water (> 95%) was obtained from
Spectra Stable Isotopes. Bis(succinimidyl)-3azidomethyl-glutarate (BAMG) and purified
Gas2p were kindly provided by Luitzen de Jong
[4,9]
. The amino acid sequences of cytochrome c
and Gas2p used in this research can be found in
the Supporting Information. All chemicals used
were either research grade or of the highest
purity commercially available.
Cross-linking and digesting cytochrome c
and Gas2p. A solution of cross-linker
bis(succinimidyl)-3-azidomethyl-glutarate
(BAMG), 20 mM dissolved in DMF, was added
to 10 µM Gas2p and 40 µM cytochrome c,
respectively, to a final concentration of 0.1 mM
BAMG (0.5% v/v DMF) in 50 mM sodium
phosphate buffer (pH 7.5) and 100 mM NaCl.
The reaction mixture was incubated for 30 min at
room temperature.
Subsequently the pH was raised to pH 9 by
addition of sodium carbonate and incubated for
another 30 min in order to quench the reaction by
saponifying unreacted N-hydroxy succinimidyl
esters and scavenge any esters that may have
formed from the reaction of cross-linker with
Ser, Tyr and Thr side chains. The remaining
reaction mixture was concentrated to 50 μl by
Biomax cutoff filter (5 kDa and 30 kDa for
cytochrome c and Gas2p, respectively) and
washed twice by phosphate/NaCl buffer.
Subsequently the proteins were denatured by
washing with 9 M Urea/50 mM citrate (pH 3)
and incubating for 10 min. Cysteines were
reduced by incubating with TCEP (final
concentration 10 mM) for 20 min at room
temperature and alkylated by incubating with
IAA (final concentration 9 M Urea, 0.2 M IAA,
0.2 M ammonium bicarbonate) for 60 min in the
dark. The protein solution was then washed four
times with a solution of 9 M Urea/50 mM
phosphate (pH 7.5) and diluted to 1 M Urea/50
mM phosphate by washing with 50 mM
phosphate. Subsequently trypsin (1:25 w/w) was
added for digestion overnight at 37 °C followed
by digestion by Glu-C overnight at 25 °C.
Samples containing 5 μg peptides were taken for
MALDI-TOF analysis.
Measurement of the pH dependency of the
Carboxyl
Oxygen
Exchange
Reaction
catalysed by Glu-C. The efficiency of carboxyl
oxygen exchange reaction catalysed by Glu-C
was determined by MALDI-TOF analysis of 18O
2
incorporation into the C-terminus of a bovine
insulin fragment. For the pH studies, a 40 μM
solution of insulin digest in aqueous buffers at
various pHs was lyophilised and reconstituted
with a 400 nM Glu-C solution in a total volume
of 10 μl 95% H218O after which it was incubated
at 25 °C. The buffer solutions used were 100
mM phosphate at pH 5.8, 6.2, 6.8, 7.4 and 7.8
and 100 mM citric acid/phosphate at pH 4, 5 and
6. The duration of the incubation was 2 hours
and was stopped by the addition of one volume
of 4% formic acid/0.2% TFA in water. The final
percentage of 18O incorporation was determined
by measuring relative heights of the 12C
monoisotopic peaks containing two, one or no
18
O atoms. The influence of the second 13C
isotope peak on 18O containing peaks is assumed
to be negligible due to the low mass of the
peptide. Ionisation efficiency is assumed not to
be affected by 18O incorporation, this leads to the
following formula:
18
O1  2*18 O2
18
Oinc 
*100%
2
In which 18O1 and 18O2 represent the relative
height of peaks corresponding to peptides with
one 18O atom or two 18O atoms, respectively.
The contribution of unlabelled peptides is
implied in that it subtracts from 18O1 and 18O2
peaks, which results a lower incorporation. The
theoretical maximum is 18O1=0 and 18O2=1
which results in 100% incorporation.
18O-labelling
of
BAMG-treated
cytochrome c and Gas2p digests. A solution of
30 µM cytochrome c and 6 µM Gas2p,
respectively, in 100 mM sodium phosphate
buffer (pH 6.2) was lyophilised and reconstituted
with a solution of 2.1 µM and 1.5 µM trypsin,
respectively, in a total volume of 10 µl H218O.
The mixture was incubated for 2 hours at 37 °C,
after which Glu-C (final concentration 2.1 µM
and 1.5 µM for cytochrome c and Gas2p,
respectively) was added and incubation resumed
at 25 °C overnight. Incubation was stopped by
addition one volume of 4% formic acid/0.2%
TFA in water.
BAMG
capture
on
cyclooctynefunctionalised beads. To purify BAMG-linked
peptides a solution of 100 µg Gas2p and 18 µg
cytochrome c, respectively, in 60 µl 50%
acetonitrile/50% 50mM potassium phosphate
buffer (pH 7.5) was prepared from previously
cross-linked and digested Gas2p and cytochrome
c. Subsequently it was added to approximately 2
mg of dry PL-DMA beads and incubated on a
turning rotavap at 40 °C for 24 hours. The
reaction was stopped by spinning down the
suspension at 12,000 g for 1 minute after which
the supernatant was collected by pipette and
stored for future reference. The supernatant from
the first washing step was added to the
supernatant removed prior to washing. This
combined fraction contains the majority of
unmodified peptides.
The beads were washed seven times: with a
solution of 60 µl 50% acetonitrile/50% 50 mM
potassium phosphate buffer (pH 7.5, twice),
100% acetonitrile, 50% acetonitrile/50% 50 mM
potassium phosphate buffer (pH 7.5), 50 mM
potassium phosphate buffer (pH 7.5), 2 M NaCl
and finally with 50 mM potassium phosphate
buffer (pH 7.5). Each washing step consisted of
turning on a rotavap for 15 min at room
temperature and spinning down at 12,000 g for 1
min after which the supernatant was removed by
pipette.
To release the captured peptides, the washed
beads were incubated with a solution of 5 mM
TCEP in 50 mM potassium phosphate buffer (pH
7.5) on a turning rotavap at room temperature for
60 min followed with 2.5 mM TCEP in 50%
acetonitrile/50% 50 mM potassium phosphate
buffer (pH 7.5) for 15 min at room temperature.
After each step, the beads were spun down and
the liquid was collected and combined by
pipette. Finally, 250 mM IAA in 50 mM
potassium phosphate (pH 7.5) was added to a
final concentration of 55 mM IAA and incubated
for 30 min at room temperature in the dark. The
resulting mixtures were cleaned by ZipTip µC18
pipette tips as per the manufacturer instructions
(Millipore, Bedford, USA).
Mass spectrometry. Peptides were collected
on ZipTip μC18 pipette tips, washed with 0.1%
TFA and eluted with 50% acetonitrile/0.1%
3
pH dependency of the Carboxyl Oxygen
Exchange Reaction for Oxidised Insulin
Digest catalysed by Glu-C. The pH dependency
and 18O incorporation over time of the carboxyl
oxygen exchange reaction is shown in figure 1a
and b, respectively.
A
18O Incorporation (%)
100
90
80
70
60
50
40
30
20
10
0
5.5
6
6.5
7
7.5
8
pH
B
100
90
18O Incorporation (%)
TFA. Mass spectrometry was performed by
MALDI-TOF, LC-ESI-Q-TOF and LC-ESIFTICR. For MALDI-TOF, 0.5 µl samples were
mixed with an equal volume of a 10 mg/ml αcyano-4–hydroxycinnaminic acid solution in
50% acetonitrile/50% ethanol. The mixture was
spotted on a MALDI target plate and allowed to
dry. MALDI-TOF spectra were acquired on a
TofSpec 2EC mass spectrometer (Micromass,
Wythenshawe, U.K.) in reflectron mode and
externally mass calibrated using a standard
peptide mixture.
For MS/MS analysis samples were diluted to
<5% acetonitrile/0.1% TFA and loaded onto a
precolumn of an Ultimate nano-HPLC system
(LC Packings, Amsterdam, The Netherlands)
and separated on a PepMap C18 nano-reversedphase column (75 µm i.d.). Elution was
performed using a gradient of 5-50% acetonitrile
with 0.1% TFA. The flow was infused directly
into an ESI-QTOF mass spectrometer
(Micromass) via a modified nanoelectrospray
device (New Objective, Woburn, MA). Argon
was used as a collision gas at 4 x 10-5 bar
measured at the quadrupole pressure gauge.
External mass calibration was done using a
standard tryptic cytochrome c digest.
Accurate mass was determined by an Apex Q
FTICR mass spectrometer (Bruker Daltonics,
Billerica, MA, USA) coupled in-line to an HPLC
equipped with an ESI ion source for which
samples were dried in a vacuum centrifuge and
reconstituted in 0.1% TFA. Mass spectra were
internally calibrated using exact masses of
known unmodified peptides.
Mass
spectra
were
analysed
with
VIRTUALMSLAB to identify unmodified (linear)
peptides, mono-link, loop-link and cross-link
candidates. VIRTUALMSLAB can perform in silico
digestions to create mass spectrometry reference
spectra and match that to mass spectrometry data
from the real experiment with expected mass
shifts from modifications.
80
70
60
50
40
30
20
10
0
0
4
8
12
16
20
24
Tim e (h)
Figure 1. Effect of pH on the carboxyl oxygen exchange
activity of Glu-C (a) and 18O incorporation followed in
time (b). Efficiency is calculated from relative peak
intensities (see materials and methods). a) A 40 µM
solution of bovine insulin (reduced, alkylated and digested
with Glu-C) was incubated with 400 nM Glu-C (1:100
molar ratio) in 100 mM sodium phosphate buffered at
various pH pHs. Incubation time was 2 hours. b) -●-:
1:100 molar ratio in sodium phosphate buffer pH 6.2. -■-:
1:20 molar ratio in sodium phosphate buffer pH 6.2.
Results
4
The pH dependency of the carboxyl oxygen
exchange reaction catalysed by Glu-C was found
to have an optimum in sodium phosphate buffer
at pH 6.2. This is 1 unit lower than the reported
optimum for amidase activity at pH 7.2[10]. The
pH 4 to 6 range in citric acid/phosphate buffer
showed a significantly lower efficiency (data not
shown). The optimum found for Glu-C is
conveniently close to the reported optimum of
pH 6 for the reaction catalysed by trypsin[11].
This facilitates a double labelling experiment
without the need for buffer adjustments.
Cross-links in cytochrome c and Gas2p.
Cross-linking was done with bis(succinimidyl)3-azidomethyl-glutarate (BAMG) by the
formation of amide bonds with the amine group
of the lysine side chain. Amine reactive crosslinkers are often used for protein cross-linking
due to the presence of multiple lysine residues on
the surface of most soluble proteins.
Table 1. Overview of cross-link candidates from cytochrome c and the observed shift after labelling.
18
Experimental [M+H]+
Calculated [M+H]+
Error (ppm)[a]
Sequence[b]
O Shift (amu)[c,d]
931.53521
931.53598
1
K8-K13 (ML)
4
975.52484
975.52581
1
K73-K79 (ML)
4
1060.54257
1060.54219
0
Y67-K73 (ML)
4
1098.64089
1098.64184
1
G6-K13 (LL)
4
1186.67616
1186.67651
0
M80-K88 (LL)
4
1261.67880
1261.67868
0
E92-K100 (ML)
4
1400.75224
1400.74922
2
K100-E104~V3-K8 (XL)
8
1602.82478
1602.82479
0
H26-R38 (ML)
Not Found
1658.83608
1658.83843
1
E92-E104 (LL)
Not Found
1701.89490
1701.89589
1
Y67-K79 (LL)
4
1767.82832
1767.82966
1
K39-K53 (ML)
Not Found
1802.98964
1802.99118
1
K73-K79~N54-K60 (XL)
4
1826.95136
1826.95211
0
G23-R38 (LL)
4
1861.02163
1861.01779
2
K5-K7~D93-E104 (XL)
1861.02163
1861.01779
2
G6-K8~D93-E104 (XL)
Not Found
1861.02163
1861.02182
0
L94-K100~G56-E62 (XL)
1864.05187
1864.05134
0
Y74-K88 (ML)
1864.05187
1864.05134
0
K73-K87 (ML)
Not Found
1864.05134
0
K87-K88~Y74-K86 (XL)
1864.05187
1864.05134
0
K73-K79~M80-K87 (XL)
1881.87191
1881.87259
0
T40-K55 (ML)
Not Found
1949.06764
1949.06772
0
Y67-K73~M80-K87 (XL)
8
1976.04754
1976.04876
1
G56-K62~D93-K100 (XL)
Not Found
1991.95540
1991.95699
1
K39-K55 (LL)
4**
2105.09018
2105.09135
1
G56-E62~E92-K100 (XL)
8
2449.18704
2449.18951
1
T40-K60 (LL)
Not Found
2480.33670
2480.33702
0
Y67-K86 (ML)
Not Found
2480.33702
0
Y67-K73~Y74-K86 (XL)
2595.29347
2595.29503
1
K39-K60 (ML)
2595.29503
1
N54-K60~K39-K53 (XL)
8*
2595.29347
2595.29503
1
A51-K60~K39-D50 (XL)
2611.24368
2611.24233
1
G56-E62~K39-K53 (XL)
8*
[a] Numbers in blue represent a mass surplus whereas numbers in red a mass deficit. [b] ML, mono-link; LL, loop-link;
XL, cross-link [c] Shifts marked with an asterisk are observed in low abundance. [d] Shifts marked with ** show very
poor 18O incorporation.
5
Table 2. Overview of cross-link candidates from Gas2p and the observed shift after labelling.
18
Experimental [M+H]+
Calculated [M+H]+
Error (ppm)[a]
Sequence Matched[b]
O Shift (amu)
928.53682
928.536313 (LL)
1
F308-K313 (LL)
4
1378.65475
1378.653214 (ML)
1
L430-R439 (ML)
4
1450.62924
1450.630748 (ML)
1
Y389-D398 (ML)
0
1507.73162
1507.732193 (ML)
0
S462-R472 (ML)
4
1539.72635
1539.728548 (XL)
1
K313-E314~A352-D361 (XL)
4
1647.72645
1647.727898 (ML)
1
V397-E410 (ML)
4
1677.807861
(XL)
2
K313-E314~I402-E413
(XL)
1677.81172
4
1677.81172
1677.807861 (XL)
2
T236-E238~N289-D298 (XL)
1767.90105
1767.898407 (XL)
1
E234-E238~K355-R362 (XL)
4
1850.96391
1850.964695 (XL)
0
F308-K312~Y328-E336 (XL)
4
1904.95190
1904.95348 (ML)
1
T52-R66 (ML)
4
1931.87003
1931.870479 (ML)
0
H440-E453 (ML)
4
1958.03384
1958.034172 (XL)
0
K7-K12~N289-D298 (XL)
Not Found
2131.99714
2131.989075 (XL)
4
S383-E388~A352-R362 (XL)
4
2209.12233
2209.124779 (LL)
1
I296-K313 (LL)
4
2287.09895
2287.091748 (XL)
3
A172-E174~T236-E250 (XL)
4
2356.177938 (ML)
0
I296-E314 (ML)
2356.17786
2356.177938 (XL)
0
K313-E314~I296-K312 (XL)
4
2391.07045
2391.059148 (XL)
5
D171-E174~V81-E95 (XL)
4
2409.07788
2409.084968 (ML)
3
Y389-K407 (ML)
4
2485.235787 (XL)
4
F308-E314~I13-E25 (XL)
2485.24643
Not Found
2485.24643
2485.257786 (XL)
5
S462-D469~L423-K433 (XL)
2549.23417
2549.237913 (XL)
1
Y481-K490~V81-D91 (XL)
4
2625.16694
2625.167452 (XL)
0
S383-E388~H440-E453 (XL)
Not Found
2641.21699
2641.217379 (XL)
0
L430-R439~Y389-D398 (XL)
Not Found
[a] Numbers in blue represent a mass surplus whereas numbers in red a mass deficit. [b] ML, mono-link; LL, looplink; XL, cross-link.
Cytochrome c is a protein with a relative large
content of lysine residues. Another useful
property of BAMG is the aptly positioned azido
group which can be used to purify peptide
mixtures.
After cross-linking and proteolytic digestion,
peptide mixtures will contain multiple crosslinker-modified peptides in addition to a vast
majority of unmodified peptides: a cross-link
within the same peptide (loop-link), a cross-link
between different peptides (cross-link) and
peptides modified by partially hydrolysed crosslinker (mono-link). A modification adds 151.038
Da in case of a cross-link or loop-link and
169.049 Da in case of a mono-link. MALDITOF mass spectrometry was used to quickly
assess whether any BAMG-linked peptides were
present.
Of the most abundant BAMG-linked
peptides, satellite signals at Δ = -26 to -28 Da
from the main peak can be found in MALDI-Tof
mass spectra (Figure 3a) which is probably due
to in-source loss of N2 from the azido group and
subsequent uptake of either two, one or no
hydrogen atoms. Accurate mass was determined
by FTICR mass spectrometry. FTICR data was
calibrated and matched to virtual mass spectra in
VIRTUALMSLAB. In order to confirm a cross-link,
the 18O incorporation experiment as described in
materials and methods was done. A cross-link
can incorporate four 18O atoms whereas a monoor loop-link can only incorporate two. This leads
6
to a difference in mass-shift and provides a
simple method to distinguish between actual
cross-links and mono- or loop-links. A typical
result of a peptide displaying a shift of 4 amu
from incorporation of two 18O atoms is shown in
Figure 2.
Figure 2. MALDI-TOF mass spectra before (A) and after
(B) 18O labelling of a cross-link candidate from Gas2p,
matched sequences are K313-E314~I402-E413 and T236E238~N289-D298. High 18O incorporation is shown but
the shift of 4 amu indicates it is a false positive.
Unfortunately, deconvoluting FTICR mass
spectra of 18O labelled peptides proved to be
impossible with the available software. Based on
the retention time of unlabelled peptides, crosslink candidates matched in VIRTUALMSLAB were
found manually. A list of cross-link candidates
from cytochrome c is shown in Table 1.
Five out of ten cross-link candidates could be
confirmed by a shift of 8 amu of which four are
consistent with previously published data by L.
de Jong et al[4]. For cross-links that could not be
confirmed by a shift of 8 amu, three no longer
appeared in the mass spectrum after labelling
whereas one candidate cross-link, K73K79~N54-K60 (m/z 1802.99), was confirmed as
a cross-link by L. de Jong et al. The
unambiguous 4 amu shift after labelling indicates
that either that particular C-terminus is blocked
for labelling, despite not being blocked for
cleavage, or it is a false positive. Sequence
analysis is required to elucidate the correct
interpretation.
The cross-link candidate K100-E104~V3-K8
(m/z 1400.75) that showed a shift of 8 amu but
was not confirmed by L. de Jong et al was
validated by fitting it to the known 3D structure
of cytochrome c in solution (PDB ID: 1AKK).
The distance between the Cα (12.07 Å) and Nε
(18.57 Å) atoms of linked lysine residues fit well
within spacer length of BAMG (7.5 Å) given the
usually flexible lysine side-chains.
The promising results with cytochrome c
indicated that the method works and was applied
directly to Gas2p. In Gas2p, however, no crosslink candidates found in VIRTUALMSLAB could be
confirmed by a shift of 8 amu. Four candidate
cross-links did not appear in the mass spectrum
after labelling. However, various peptides
displaying a shift of 8 amu were found (m/z
2970.36 and 2549.23, data not shown) that could
not be matched in VIRTUALMSLAB. In general,
relatively few matches could be made in
VIRTUALMSLAB considering the size and complexity
of Gas2p compared to cytochrome c. It is likely
that various possible modifications that have not
been taken into account have caused this. On the
other hand, many cross-link candidates were
proven to be false positives. With an increase in
size of a protein, and thus the amount of lysine
residues, it becomes more likely to find a mass
match for any given cross-link candidate.
Due to the low abundance of BAMG-linked
peptides,
cyclooctyne-functionalised
polydimethylacrylamide (PL-DMA) beads were used
to purify the peptide mixture. A strain-promoted
[3+2] alkyne-azide cycloaddition (clickchemistry[12]) selectively and quickly captures
BAMG-linked peptides. To release the captured
peptides after washing, a disulfide bond in the
7
spacer with which the functional group
cyclooctyne is linked to the beads is reduced and
alkylated by TCEP and IAA (see materials and
methods for details). This adds an additional
358.17 Da to BAMG-linked peptides. The
shifted MALDI-TOF mass spectrum of
cytochrome c is shown for a BAMG-linked
peptide in Figure 3. As expected, the most
abundant BAMG-linked peptide in cytochrome c
previously found at m/z 1827.13 (LL) now
appeared at m/z 2185.09 whereas the satellite
signals have disappeared. Unfortunately, the
most abundant BAMG-linked peptide from
Gas2p in the FTICR mass spectrum at m/z
928.53 had low ionisation efficiency with
MALDI.
Figure 3. MALDI-TOF mass spectra of cytochrome c
cross-linked with BAMG and digested by trypsin and GluC. a) A BAMG-modified peptide with its characteristic
satellite signals at Δ = -26 to -28 Da from the main peak by
in-source loss of N2 and uptake of two, once or no
hydrogen atoms. b) After purification with cyclooctynebeads, a corresponding 358.17 Da shift is observed and no
satellite signals remain.
The captured peptide became the most abundant
peak, however. Except for the heme-containing
peptide from cytochrome c, no unmodified
peptides were recovered from the beads in any
significant amount (data not shown). Labelling
experiments have been attempted with captured
peptides but yielded poor results. Additional
research is required to optimise conditions for
labelling after capture.
Discussion
It has been described that monovalent, planar
anions like acetate inhibit Glu-C[10]. The
conjugated base of citric acid shows a similar
inhibition, with almost no 18O incorporation at
pH 5 and 4 and significantly less incorporation at
pH 6 than in sodium phosphate buffered at pH
5.8 and 6.2. Interestingly, the citric acid buffer
also contained sodium phosphate which implies
that the inhibition by citric acid is much stronger
than the activation by phosphate. The time
course of 18O incorporation shows an outlier at
the 16 hour data point, this can be explained by
the fact that the 2, 4, 6, 8 and 24 hour samples
were taken from the same batch whereas the 16
hour sample was taken from a separate
incubation. Due to time constraints this sample
was not lyophilised overnight as normal which
could have left some remaining 16O water. The 2
hour data point shows only 65% 18O
incorporation while the pH 6.2 data point in (a)
shows nearly 80% at the same incubation
conditions (pH 6.2, 2 hours incubation). The
following data points of the same batch (4, 6 8
and 24 hours) do show higher incorporation, up
to the theoretical maximum, which suggests less
enzyme was present than calculated. This is also
supported by the 2 hour data point of the 1:20
molar ratio experiment, which already shows
complete incorporation (> 90%).
Labelling cross-linked peptides raised some
problems. Many peaks that could not be matched
by VIRTUALMSLAB did show a shift of 4 amu which
indicates they are peptides. The most likely
explanation is that these peaks belong to cross-
8
and auto-digestion of Glu-C and trypsin, despite
using modified trypsin resistant to autodigestion[9]. Trypsin is known to show
chymotrypsin-like activity when digested and it
is not fully inactive at 25 °C during incubation
with Glu-C. This leads to the conclusion that a
better protocol would be to digest with Glu-C
first, followed by incubation with trypsin at high
temperature to avoid as much cross digestion as
possible. Previous research has shown that
trypsin retains high activity up to 50 °C[13] but
whether this includes the carboxyl oxygen
exchange reaction remains to be seen. Another
possibility would be to start the exchange
reaction with Glu-C and boil the mixture before
trypsin is added in order to denature Glu-C
which does not have disulfide bridges.
The first labelling experiments with
cytochrome c and Gas2p yielded an 18O
incorporation efficiency lower than what was
expected at the enzyme concentrations used (data
not shown). The molar ratio of 1:100 is based on
the undigested protein but when digestion is
taken into account, the amount of peptides
eligible for labelling is increased significantly.
Insulin only yields four peptides per digested
molecule that can be labelled after Glu-C
digestion whereas doubly digested cytochrome c
and Gas2p yield 29 and 94 peptides,
respectively. When corrected for these
differences, 18O incorporation was as expected
for most peptides. However, labelling efficiency
varies between peptides as has been previously
described[7]. Accurate recognition of cross-links
is hampered by incomplete 18O incorporation due
to overlap with 13C isotope peaks of large
peptides, in some cases it becomes difficult to
distinguish a shift of 4 amu from a shift of 8
when there are multiple 13C isotope peaks with
similar intensity. This underlines the importance
of near complete 18O incorporation. Peptides
labelled with 18O consistently showed an
unanticipated HPLC retention time shift of up to
-30 sec compared to unlabelled peptides[5].
While BAMG-linked peptide-capturing with
cyclooctyne functionalised PL-DMA beads is an
efficient method to reduce the complexity of a
peptide mixture, several improvements have to
be made. Due to the large excess of beads,
unreacted cyclooctyne species become the
dominant fraction after release by TCEP. This, in
addition to the unknown efficiency of crosslinking and capture, makes it difficult to estimate
the concentration of peptides after purification as
it contains two amide bonds that contribute to
A214 in UV/vis. As the molecule is uncharged,
Strong Cathode Exchange (SCE) is likely to be
an efficient method of purifying the peptide
mixture. However, this adds two more
purification steps (desalting after SCE), resulting
in a poor overall yield. Therefore, using a much
higher initial concentration of peptides is
advisable.
Acknowledgement
I would like to thank Luitzen de Jong and Merel
Nessen for supervising my work, Leo de Koning
for helping me analyse the FTICR data of my
most recent samples, and last but not least,
Ronald Aardema, for putting up with my endless
stream of questions. I would like to thank
everyone at SILS-Massa that made my time here
such a pleasant experience.
References
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2.
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5.
Back, J. W., de Jong, L., Muijsers, A. O. & de
Koster, C. G. (2003) Chemical cross-linking and
mass spectrometry for protein structural
modeling, J Mol Biol. 331, 303-13.
Sinz, A. (2006) Chemical cross-linking and mass
spectrometry to map three-dimensional protein
structures and protein-protein interactions, Mass
Spectrom Rev. 25, 663-82.
A. Fonatana, G. Fassina, C. Vita, D. Dalzoppo,
M. Zamai, M. Zambonin, Biochemistry 1986, 25,
1847-1851.
Kasper, P. T., Back, J. W., Vitale, M., Hartog, A.
F., Roseboom, W., de Koning, L. J., van
Maarseveen, J. H., Muijsers, A. O., de Koster, C.
G. & de Jong, L. (2007) An aptly positioned
azido group in the spacer of a protein cross-linker
for facile mapping of lysines in close proximity,
Chembiochem. 8, 1281-92.
Back, J. W., Notenboom, V., de Koning, L. J.,
Muijsers, A. O., Sixma, T. K., de Koster, C. G. &
de Jong, L. (2002) Identification of cross-linked
9
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peptides for protein interaction studies using mass
spectrometry and 18O labeling, Anal Chem. 74,
4417-22.
Sprinson, D. B.; Rittenberg, D. Nature (London)
1951, 167, 484.
Reynolds, K. J., Yao, X. & Fenselau, C. (2002)
Proteolytic 18O labeling for comparative
proteomics: evaluation of endoprotease Glu-C as
the catalytic agent, J Proteome Res. 1, 27-33.
de Koning, L. J., Kasper, P. T., Back, J. W.,
Nessen, M. A., Vanrobaeys, F., Van Beeumen, J.,
Gherardi, E., de Koster, C. G. & de Jong, L.
(2006) Computer-assisted mass spectrometric
analysis of naturally occurring and artificially
introduced cross-links in proteins and protein
complexes, Febs J. 273, 281-91.
Popolo, L. Ragni, E. Carotti, C. Palomares, O.
Aardema, R. Back, J. W.; Dekker, H. L.; de
Koning, L. J.; de Jong, L & de Koster, C. G.
(2008) Disulfide Bond Structure and Domain
Organization of Yeast β(1,3)-
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Glucanosyltransferases Involved in Cell Wall
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Sorensen, S. B., Sorensen, T. L. & Breddam, K.
(1991) Fragmentation of proteins by S. aureus
strain V8 protease. Ammonium bicarbonate
strongly inhibits the enzyme but does not improve
the selectivity for glutamic acid, FEBS Lett. 294,
195-7.
Hajkova, D., Rao, K. C. & Miyagi, M. (2006) pH
dependency of the carboxyl oxygen exchange
reaction catalyzed by lysyl endopeptidase and
trypsin, J Proteome Res. 5, 1667-73.
Kolbet, H. C. et al. Diverse Chemical Function
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Fraser, D. Johnson, F. H. (1950) Pressuretemperature relationship in the rate of casein
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10
Supporting Information
Sequence of horse heart cytochrome c used in VIRTUALMSLAB:
1 Ac-GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP 30
31
NLHGLFGRKT GQAPGFTYTD ANKNKGITWK 60
61
EETLMEYLEN PKKYIPGTKM IFAGIKKKTE 90
91
REDLIAYLKK ATNE 104
Sequence of Gas2p used in VIRTUALMSLAB:
1
51
101
151
201
251
301
351
401
451
501
RGVSFEKTPA
ETSYIDALAD
GMYVLLDLSE
EVTNDHTNTF
ARYFVCGDVK
FGCNLVRPRP
GVDILPDFKN
EANEKLPETP
DILANGKTGE
NLESLQPLTS
EEREHHHHHH
IKIVGNKFFD
PKICLRDIPF
PDISINRENP
ASPFVKAAIR
ADFYGINMYE
FTEVSALYGN
LKKEFAKADP
DRSKCACLDE
YGEFSDCSVE
ESICKNVFDS
510
SESGEQFFIK
LKMLGVNTLR
SWDVHIFERY
DAKEYISHSN
WCGYSTYGTS
KMSSVWSGGL
KGITEEEYLT
ILPCEIVPFG
QKLSLQLSKL
IRNITYNHGD
GIAYQLQRSE
VYAIDPTKSH
KSVIDAMSSF
HRKIPVGYST
GYRERTKEFE
AYMYFEEENE
AKEPTEVESV
AESGKYEEYF
YCKIGANDRH
YSKSNPSRSK
EELSNANGAF
DICMEALSAE
PNLLGYFAGN
NDDAMTRDNL
GYPIPVFFSE
YGVVKINDND
ECPHIAVGVW
SYLCSKVDCS
CPLNDKNVYF
ESLNVKYPSS
50
100
150
200
250
300
350
400
450
500
Figure 1. MALDI-TOF mass spectrum of 18O labelled insulin peptide from which labelling efficiency was calculated, A)
Unlabelled reference, B) 2 hours incubation, C) 4 hours, D) 6 hours, E) 8 hours, F) 16 hours, G) 24 hours.
11