Higher-energy C-trap dissociation (HCD) for precise peptide ... · HCD of acetylated...
Transcript of Higher-energy C-trap dissociation (HCD) for precise peptide ... · HCD of acetylated...
Higher-energy C-trap dissociation (HCD) for precise peptide
modification analysis
Jesper V Olsen, Boris Macek, Oliver Lange, Alexander Makarov, Stevan Horning & Matthias Mann
Supplementary figures and text:
Supplementary Figure 1 Two HCD configurations.
Supplementary Figure 2 HCD of acetylated lysine-containing peptides.
Supplementary Figure 3 Determination of y-ions for de novo sequencing.
Supplementary Table 1 Identified phosphotyrosine-containing peptides.
Supplementary Methods
Supplementary Figure 1 - Two HCD configurations
Potential
x
Gas <1 mtorr
Linear ion trap (LTQ) C- trap
Orbitrap
ESI source
(i) (ii) (iii)
(i) (ii) (iii)a LTQ Orbitrap - HCD with fragmentation in C-trap
b LTQ Orbitrap XL - HCD with fragmentation in octopole collision cell
C-Trap II OctopoleCollision Cell
-
LTQ
Orbitrap
ESI
Oct 2 DC Offset =-„HCD“ CE
(-10..250 V)
Stage 1. HCD in octopole collision cell(10...20 ms duration)
+6V
0 +6V
Stage 2.Transfer fragment ions to the C-trap (10...30 ms duration) 0
-(- -
0
0 +15V
OctopoleCollision Cell
C-Trap II LTQ
Description of the two HCD configurations: The figure shows two configurations used for Higher Energy Dissociation (HCD) on the linear ion trap orbitrap (LTQ-Orbitrap) hybrid instrument. The first implementation, depicted in panel a, uses the C-trap to fragment the ions that are first isolated in the linear ion trap part of the instrument. The inset at the top of the panel shows the potential energy diagram and the gas pressure in the C-trap. In the second iteration, depicted in panel b, an octopole collision cell is added to the instrument. It has the following specifications: The octopole collision cell (120 mm length, 5.5 mm id, 2 mm rod diameter) is enclosed in a gas tight shroud is aligned to the C-trap device. The collision cell is supplied with a RF voltage (2.6 MHz, 500 V p-p) of which the DC offset can be varied ±250 V and a collision gas of choice (usually nitrogen). Higher energy collisions (HCD) take place as follows (see potential energy diagram at the top of panel b): Ions of a determined number, either mass selected or not, are transferred from the linear ion trap to the C-trap. The C-trap is held at ground potential. For HCD, ions are emitted from the C-trap to the octopole by setting a trap lens. Ions collide with the gas in the octopole at an energy which is determined as a relative energy depending on the ion mass, charge, and also the nature of the collision gas (normalized collision energy). Thereafter, the product ions are transferred from the octopole back to the C-trap by raising the potential of the octopole. A short time delay (30 ms) is used to ensure that all of the ions are transferred. In the final step, ions are ejected from the C-trap into the orbitrap analyzer. Advantages of the octopole configuration over the C-trap configuration The c-trap is a curved liner ion trap. Under normal operating conditions the ions to be trapped have a kinetic energy of a few eV, and trapping efficiency is very high for all m/z. If the C-trap is used as a collision cell, then the curvature imposes a limitation. At high kinetic energies, incoming high mass ions will be lost, unless the RF amplitude on the rods is increased significantly. Increasing the RF amplitude, however, changes the low mass cut-off point for fragment ion storage to higher m/z values. Therefore C-trap fragmentation requires a compromise setting of the RF amplitude, just sufficient to not lose high mass ions at a given collision energy but accepting a change in the low mass cut-off mass for stored fragment ions. In the octopole collision cell such a compromise setting is not required. In addition, the collision gas pressure and the nature of the collision gas can be selected freely in the octopole collision cell for best fragmentation performance. This is not the case in the c-trap where increasing the gas pressure will also increase the pressure in the orbitrap analyzer.
Im(ε-ac-K) Theoretical m/z = 126.0919:
Supplementary Figure 2 - HCD of acetylated lysine-containing peptides
CH
NHCH2
CH2
CH2
CH2H2N
C
O
H3C
+
AcLys immonium ion, m/z = 143.1184
HCD
- NH3
C
NCH2
CH2
CH2
CH2
C
O
H3C +
H
AcLys* immonium ion, m/z = 126.0919
0 5 10 15 20 25 30 35 40 45 50Time (min)
0
20
40
60
80
100
Rel
ativ
e ab
un
dan
ce
37.07
40.66
SIC of m/z 126.091-126.093
NL: 6.36E3
L G L acK S L V S KHistone H1 preparation
?
HCD of acetylated lysine-containing Histone H1 peptide. (a) A peptide from linker histone H1 was fragmented by HCD. The inset shows the immonium ion characteristic of acetylated lysine (m/z = 126.0919). (b) Selected ion chromatogram of the reporter ion for acetylated lysine from a histone H1 preparation. The figure depicts the extracted signal for the mass range 126.091-126.093 of every HCD MS/MS spectrum in the LC run. The main peak is due to an acetylated peptide. (c) The structure and generation of the reporter ion is shown. The chemical composition of this reporter ion is not completely unique to acetylated lysine and the second peak in the extracted ion chromatogram, labeled with a question mark, may be due to a non-acetylated peptide.
c
100 300 500 700 900m/z
HCD-MS/MS
L G L acK S L V S K
L
y1
y72+
y8
y7
y6y5
MH22+y2
y3b3b2
Histone H1 lysine acetylation
b8b7b6b5
b4y4
120 122 124 126 128 130
126.0918
20
40
60
80
100
Rel
ativ
e ab
un
dan
ce
0
a129.1027
acK
b
a bMASCOT ion score: 227MASCOT ion score: 161
Supplementary Figure 3 - Determination of y-ions for de novo sequencing
A SILAC double-labeled peptide (Fig. 3c,d) was isolated with a broad mass window and both SILAC states fragmented together by HCD using the collision octopole in Fig. 1b. (a) Database search results using just the y-ion data extracted from Fig .3d with the Mascot algorithm. (b) Database search results for the complete series of calculated y-ions.
Supplementary Table 1 – Identified Phosphotyrosine‐containing peptides Retention Time [min]
216.043?
Swiss‐Prot or TrEMBL accession
Protein name
Phosphopeptide sequence Modifi‐cations
z Mascot score
Mass error [ppm]
36.18 yes P98179 RNA‐binding protein 3
YSGGNYRDNpYDN 1pSTY 2 25 ‐0.41
36.30 yes Q16539‐1 p38 MAPK
HTDDEMoxTGpYVATR 1Arg6 1Met(ox) 1pSTY
2 46 ‐1.22
36.34 yes Q16539‐1 p38 MAPK
HTDDEMoxTGpYVATR 1Met(ox) 1pSTY
2 42 ‐0.72
38.58 no P12931‐2 Tyrosine kinase Src
LIEDNEpYTAR 1Arg10 1pSTY
2 35 ‐0.06
39.28 yes P00533‐1 EGF receptor
GSTAENAEpYLR 1Arg6 1pSTY
2 69 0.33
39.34 yes P00533‐1 EGF receptor
GSTAENAEpYLR 1pSTY 2 56 0.29
39.78 yes Q8TF42 STS‐1 ac‐AAREELpYSK 1pSTY N‐ac
2 41 0.25
40.53 yes P49023‐2 Paxiilin FIHQQPQSSpSPVpYGSSAK 2pSTY 3 22 0.48
41.17 yes Q8N3N5;Q86YV5
Tyrosine kinase FLJ00269
EATQPEPIpYAESTK 1Lys4 1pSTY
2 21 ‐0.45
43.14 yes Q16539‐1 p38 MAPK
HTDDEMTGpYVATR 1pSTY 2 81 ‐0.65
43.23 yes Q16539‐1 p38 MAPK
HTDDEMTGpYVATR 1Arg6 1pSTY
2 83 ‐0.63
47.59 yes Q8IVM0‐1 Ymer (c3orf6)
AYADSpYYYEDGGMoxKPR 1Met(ox) 1pSTY
3 30 ‐0.26
47.98 yes O15357 SHIP‐2 TLSEVDpYAPAGPAR 1pSTY 2 62 ‐1.04
48.05 yes O15357 SHIP‐2 TLSEVDpYAPAGPAR 1Arg6 1pSTY
2 42 ‐2.00
48.27 yes Q9NQC7‐1 CYLD VTSPpYWEER 1pSTY 2 25 ‐0.64
48.42 yes P49023‐2 Paxillin VGEEEHVpYSFPNK 1pSTY 2 59 0.67
48.59 yes P49023‐2 Paxillin VGEEEHVpYSFPNK 1Lys8 1pSTY
2 23 0.19
48.97 yes P06493 CDK1 IGEGTpYGVVYK 1Lys8 1pSTY
2 63 0.71
49.00 yes P06493 CDK1 IGEGTpYGVVYK 1pSTY 2 47 0.54
50.62 yes Q5T185 Shc PSpYVNVQNLDK 1pSTY 2 60 ‐0.23
50.63 yes Q5T185 Shc PSpYVNVQNLDK 1Lys4 1pSTY
2 42 ‐1.50
54.13 yes Q8IVM0‐1 Ymer (c3orf6)
AYADSYpYYEDGGMKPR 1pSTY 3 62 ‐0.10
56.35 yes P19174 PLC gamma
IGTAEPDpYGALYEGR 1Arg6 1pSTY
2 55 2.25
56.41 yes P19174 PLC gamma
IGTAEPDpYGALYEGR 1pSTY 2 61 2.30
57.52 yes O75886‐1 STAM2 SLpYPSSEIQLNNK 1Lys4 1pSTY
2 78 ‐0.08
63.73 yes Q9NZV1 CRIM‐1 QNHLQADNFpYQTV 1pSTY 1pyro
2 10 0.94
64.21 yes P19174 PLC gamma
pYQQPFEDFR 1pSTY 2 41 1.24
66.35 yes Q99704‐1 DOK1 IAPCPSQDSLpYSDPLDSTSAQAGEGVQR
1pSTY 3 35 ‐0.91
67.07 yes O60784 TOM1 EVKpYEAPQATDGLAGALDAR 1Arg6 1Lys4 1pSTY
3 27 0.33
Supplementary Table 1 – Identified Phosphotyrosine‐containing peptides 67.17 yes O60784 TOM1 EVKpYEAPQATDGLAGALDAR 1pSTY 3 22 0.58
68.54 yes P28482 ERK2 VADPDHDHTGFLpTEpYVATR 1Arg6 2pSTY
3 28 ‐0.55
68.69 yes P28482 ERK2 VADPDHDHTGFLpTpYVATR 2pSTY 3 19 ‐1.00
68.81 yes Q5T9K6 c10orf45 ac‐AEPDpYIEDDNPELIRPQK 1pSTY 1N‐ac
2 38 ‐0.60
70.00 yes P50402 Emerin GYNDDpYYEESYFTTR 1pSTY 2 59 ‐0.16
70.32 yes P28482 ERK2 VADPDHDHTGFLTEpYVATR 1pSTY 3 96 0.39
70.65 yes P28482 ERK2 VADPDHDHTGFLTEpYVATR 1Arg6 1pSTY
3 78 ‐0.36
73.53 yes P27361 ERK1 IADPEHDHTGFLTEpYVATR 1Arg6 1pSTY
3 43 0.32
73.86 yes P27361 ERK1 IADPEHDHTGFLTEpYVATR 1pSTY 3 39 ‐0.33
76.28 yes P00533‐1 EGF receptor
GSTAENAEpYLRVAPQSSEFIGA 1pSTY 3 35 1.64
76.69 yes Q6IPQ2;Q8IZW7
Tensin 3 protein
LSLGQpYDNDAGGQLPFSK 1pSTY 2 50 1.90
78.54 yes P00533‐1 EGF receptor
GSHQISLDNPDpYQQDFFPK 1pSTY 3 56 ‐0.33
81.35 yes P04083 Annexin A1
QAWFIENEEQEpYVQTVK 1pSTY 3 67 ‐0.89
81.88 yes Q5T185 Shc ELFDDPSpYVNVQNLDK 1pSTY 2 94 1.21
88.42 yes Q5T185 Shc QMoxPPPPPCPGRELFDDPSpYVNVQNLDK
1Met(ox) 1pSTY 1pyro
3 42 ‐0.65
89.62 yes O14964 Hrs AEPMoxPSASSAPPASSLpYSSPVNSSAPLAEDIDPELAR
1Arg6 1Met(ox) 1pSTY
3 28 0.40
89.67 yes H‐INV:HIT000015803
Eps15 EADPSNFANFSApYPSEEDMIEWAK 1Met(ox) 1pSTY
3 26 1.06
91.09 yes Q96P48‐3 Centaurin delta 2
LFPEFDDSDpYDEVPEEGPGAPAR 1pSTY 3 32 ‐0.62
92.03 yes P98082‐1 Disabled homolog 2
DSFGSSQASVASSQPVSSEMpYRDPFGNPFA
1Met(ox) 1pSTY
3 41 ‐0.20
93.07 yes O14964 Hrs AEPMPSASSAPPASSLpYSSPVNSSAPLAEDIDPELAR
1Arg6 1pSTY
3 40 ‐1.69
95.11 yes P04626 ErbB‐2 GTPTAENPEpYLGLDVPV 1pSTY 2 46 ‐1.24
97.09 yes Q6IBN9;Q53FQ5;Q13137;Q53HB5;Q9BTF7
NDP52 LLSYMGLDFNSLPpYQVPTSDEGGAR 1Met(ox) 1pSTY
3 27 ‐0.97
98.63 yes O00560 Syntenin‐1
LpYPELSQYMGLSLNEEEIR 1Arg6 1pSTY
3 59 ‐0.85
98.75 yes O00560 Syntenin‐1
LpYPELSQYMGLSLNEEEIR 1pSTY 3 15 ‐1.73
100.42
yes P04083 Annexin A1
pyroQAWFIENEEQEpYVQTVK 1pSTY 1pyro
2 84 ‐0.60
100.49
yes P98082‐1 Disabled homolog 2
ENSSSSSTPLSNGPLNGDVDpYFGQQFDQISNR
1pSTY 3 77 0.24
Supplementary text, material and methods for Olsen et al. HCD Materials and Methods Parameters and methods for C-trap fragmentation
Experiments were carried out on a standard LTQ Orbitrap instrument (4) under Xcalibur 2.0 with LTQ Orbitrap Tune Plus Developers Kit version 2.0 software. The HCD normalized collision energy is set by the user in the Instrument Method. The amplitude of the C-trap RF is determined automatically but can be adjusted slightly by increasing or decreasing the value of Activation Q in the Instrument Method.
Typical mass spectrometric conditions were: spray voltage, 2.4 kV; no sheath and auxiliary gas flow; heated capillary temperature, 150ºC; normalized CID collision energy 35% for MS2 in LTQ and 55V at m/z 1000 and scaled with m/z for HCD (C-trap CID). The ion selection threshold was 500 counts for MS2. An activation q = 0.25 and activation time of 30 ms were used.
SILAC encoded HeLa Phosphotyrosine peptides. Anti-phosphotyrosine immunoprecipitation of EGF-stimulated SILAC 1 encoded HeLa cells was performed as previously described 2 with a few modifications. Serum starved HeLa cells labeled with either L-arginine and L-lysine, L-arginine-U-13C614N4 and L-lysine-2H4 or L-arginine-U-13C6-15N4 and L-lysine-U-13C6-15N2 (2 15 cm dishes per condition; ca. 4 x 107 cells of which about a quarter was used in each experiment; ~ 95% confluent cells) were treated with 150 ng/ml of EGF (3.3 μl of 1μg/μl stock solution) for 0 min, 5 min and 10 min, respectively. Another set of SILAC encoded HeLa cells were treated with EGF for 1, 5 and 20 min respectively [ref. Olsen et al, Cell 2006]. In a second experiment, double-triple SILAC encoded HeLa cells were treated with 150 ng/ml of EGF (3.3 μl of 1μg/μl stock solution) for 0 min, 5 min and 5 min with 20 min preincubation with ortho-vanadate, respectively.
All media were removed and the cells were then lysed in modified RIPA buffer containing 1% NP-40, 0.1% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 7.5, 1 mM sodium ortho-vanadate, 5mM NaF, 5 mM β-glycerophosphate and protease inhibitors (Complete tablets, Roche Diagnostics) and left on ice for 15 minutes.
The cells were scraped, collected and then vortexed for 2 minutes. The lysates were mixed 1:1:1 then centrifuged at 17,000g (12,000 rpm Sorval SS-34) for 15 minutes to pellet cellular debris.
The lysates (supernatant) were pre-cleaned on 800 μl protein A beads for 1 hr before incubation with 1 ml (50% slurry) agarose-conjugated anti-phosphotyrosine antibody 4G10 and 150 μl agarose-conjugated anti-phosphotyrosine P-Tyr-100 for an additional 4 hrs.
Precipitated complexes were then washed with lysis buffer and PBS, and subsequently eluted with 8 M urea in 1% N-octyl glycoside pH 6.5. The eluted proteins were reduced for 20 minutes at RT in 1 mM dithiothreitol (DTT) and then alkylated for 15 minutes by 5.5 mM iodoacetamide (IAA).
Endoproteinase Lys-C (Wako) was added and the lysates were digested over night at RT. The resulting peptide mixtures were diluted 4-fold with 10 mM Tris, pH 8.0 to achieve a final urea concentration below 2M. Further treatement was essentially as in 3.
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Finally, phosphopeptides were enriched using titansphere (GL Sciences) chromatography as described 4,5
In-solution digestion of BSA. 1.0 mg of lyophilized bovine serum albumin (BSA, Sigma-Aldrich) was resolubilized in a buffer containing 6 M urea (Invitrogen, Carlsbad CA), 2 M thiourea (Fluka, Switzerland) and reduced, alkylated and digested essentially as described in6. Disulfide bonds were reduced in 10 mM for 45 min and subsequently alkylated with iodoacetamide (IAA, 50 mM final conc.) for 30 minutes at room temperature.
The reduced and alkylated BSA proteins were digested as described previously 3. Proteolysis was quenched by acidification of the reaction mixtures with glacial acetic acid. Finally, the resulting peptide mixtures were desalted on RP-C18 StageTips as previously described7 and diluted in 0.1% TFA for nanoLC-MS/MS analysis. NanoLC-MS/MS and data analysis. All nanoLC-MS/MS-experiments were performed on a Agilent Technologies 1100 nanoflow system connected to an LTQ-Orbitrap (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark) as described 3 with a few modifications. Briefly, the mass spectrometer was operated in the data dependent mode to automatically switch between MS and MS/MS acquisition. Survey full scan MS spectra (from m/z 300 – 2000) were acquired in the orbitrap with resolution R=60,000 at m/z 400 (after accumulation to a ‘target value’ of 1,000,000 in the linear ion trap). The five most intense ions were sequentially isolated and fragmented in the linear ion trap using collisionally induced dissociation (CID) at a target value of 100,000 or fragmented in the C-trap by higher-energy CID with a target value of 50,000 to 100,000. For all measurements with the orbitrap detector a lock-mass ion from ambient air (m/z 429.08875) was used for internal calibration as described in 3.
Peptides and proteins were identified via automated database searching (Matrix Science, London, UK) of all tandem mass spectra against an in-house curated target/decoy database (forward and reversed version of the human International Protein Index protein sequence database (IPI, versions 3.13, 114096 protein sequences, EBI, http://www.ebi.ac.uk/IPI/)) containing all mouse protein entries from Swiss-Prot, TrEMBL, RefSeq and Ensembl as well as frequently observed contaminants. Spectra were normally searched with a mass tolerance of 5 ppm in MS mode and 0.01 Da in MS/MS mode and strict trypsin specificity. 1 S. E. Ong, B. Blagoev, I. Kratchmarova et al., Mol Cell Proteomics 1 (5), 376
(2002). 2 B. Blagoev, S. E. Ong, I. Kratchmarova et al., Nat Biotechnol 22 (9), 1139 (2004). 3 J. V. Olsen, L. M. de Godoy, G. Li et al., Mol Cell Proteomics 4 (12), 2010
(2005). 4 M. R. Larsen, T. E. Thingholm, O. N. Jensen et al., Mol Cell Proteomics 4 (7),
873 (2005). 5 J. V. Olsen, B. Blagoev, F. Gnad et al., Cell 127 (3), 635 (2006).
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6 L. J. Foster, C. L. De Hoog, and M. Mann, Proc Natl Acad Sci U S A 100 (10), 5813 (2003).
7 J. Rappsilber, Y. Ishihama, and M. Mann, Anal Chem 75 (3), 663 (2003).
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