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.

1

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).

2

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).

3