ETD & ETD/PTR Electron Transfer Dissociation Proton Transfer Reaction Title of sales presentation – Remarks: „Bruker Confidential“ needs to be erased (slide master!) for external use. Electron Transfer Dissociation Proton Transfer Reaction Page 1 1

ETD versus CID ETD electron transfer surpasses internal heating rapid bond cleavage (no energy dissipation) random fragmentation of peptide backbone leaves labile bonds like from PTMs intact N-C bond cleavage yields c- and z-ion preferable charge state z > 2 Conventional (resonant) CID via several collisions with Helium precursor ion is internally heated preferences for weak bond cleavages nearby selected amino acids (E, D, P) backbone cleavage is preferred b- and y-ions (and internal fragments) best fragment spectra from 2+ ions y2 y3 b3 b2 b1 y1 z3 z2 z1 c1 c2 c3 Page 2

Multiply charged analyte (n≥ 2) ETD Reaction Scheme Multiply charged analyte (n≥ 2) Reagent radical anion odd-electron protonated peptide n+ (n-1)+ - + Electron- transfer Cleavage of N-Cα bond Prerequisite: multiply charged precursor ions, n ≥ 2 ! ETD is not applicable to 1+ or negatively charged ions Page 3

ETD: No Cleavage at Proline Even though the N-C bond is cleaved no respective c and z fragments are formed since they stay connected via the Proline ring system. Page 4

The “3D Advantage” Non-linear Paul Trap: Dual injection and storage of ions of both polarities peptide cations & reagent anions Cations and anions are pushed towards the center of the trap Direct ETD reaction as soon as anions enter the trap Better cross sections for ion-ion-reactions in 3D trap due to compression into the same globular volume  highly efficient ETD reaction Spec: ≥ 18 unique peptides from 5 fmol BSA on column (Easy-nLC) Page 5 Page 5

detailed characterization: preparation artefacts Use of ETD for detailed Protein Characterization Analysis of post-translational modifications (PTMs) phosphorylation glycosylation deamidation etc. Identification of sample preparation artifacts MS/MS of large peptides Combination of CID and ETD data for improved characterization of peptides and proteins, e.g. for QC applications. protein ID detailed characterization: PTM protein termini mixed modifications Protein ID is the first step in proteomics but in order to address the relevant topics a more detailed protein characterization is required. PTM analysis is an important subject because the biological activity of proteins is often determined by post-translational modifications, e.g. phosphorylation, glycosylation and so on. In the context of detailed protein characterization one needs to be able to handle multiple modifications as well as unexpected modifications. These modifications can have a natural origin or can be also preparation artefacts. For the quality control of recombinant proteins the determination of N- and C-terminus are often of great importance. Classical CID bottom-up approaches mostly fail to answer these questions whereas ETD and ETD/PTR are the perfect tools for these applications. preparation artefacts Page 6 Page 6 6

+ Strategy for phosphopeptides: PTMScanTM PTMScanTM = neutral loss triggered ETD MS Loss of H3PO4: m = 98 Combination of fast MS/MS for best sequence coverage (CID) and detailed analysis of modified peptides (ETD) CID autoMS/MS Loss of Δm/z 49, 32.6 and product ion among top N most intense MS/MS fragments ? No ! Yes ! ETD auto-MS2 of original intact PRECURSOR ion CID autoMS3 of neutral loss product ion(s) + Page 7 Page 7

PTMScanTM = Neutral Loss Triggered ETD phosphopeptide from asialo fetuin (tryptic digest) 3+ 760.6 355.1 1+ 445.1 536.8 2+ 644.3 738.4 880.9 956.4 1141.0 1287.1 303.2 440.3 844.4 932.4 1070.5 1166.7 0.0 0.5 1.0 1.5 2.0 2.5 2 4 6 200 400 600 800 1000 1200 1400 1600 m/z 728.0 3+ 722.0 Intens. x106 x104 MS 3+ 760.6 728.0 3+ Auto CID MS/MS loss of 32.6 triggers ETD MS/MS of 760.6 (3+) Page 8 Page 8 8

Phosphoscan CID versus PTMScan ETD HTFSGVASVESSSGEAFHVGK, 2x phosphorylated, MW = 2279.9 Da from asialo fetuin (tryptic digest) CID: merged MS2 & pseudoMS3 CID MSn: Phosphorylation can not be assigned Question 2: Why is everybody so excited about this? What is the motivation to use ETD and what are main applications Page 9 Page 9 9

Phosphoscan CID versus PTMScan ETD HTFSGVASVES*SS*GEAFHVGK, 2x phosphorylated, MW = 2279.9 Da from asialo fetuin (tryptic digest) ETD MS² ETD ► Phosporylation at S11 and S13 Question 2: Why is everybody so excited about this? What is the motivation to use ETD and what are main applications Page 10 Page 10 10

? Alternating CID-ETD for phosphopeptide analysis Identification of phosphorylation sites from a mixture of different caseins. ► Observation of several CID spectra showing a neutral loss of 105 Da instead of 98! Those spectra could not be identified via Mascot database search 330.6 2+ 444.2 660.2 1+ 826.3 1134.7 1331.5 1495.2 551.2 3+ MS, 11.7 min 516.3 CID (551.2) 259.1 361.2 798.3 918.3 1020.5 1146.4 1293.4 1595.7 ETD (551.2) 0.25 0.50 0.75 8 x10 Intens. 2 4 7 0.0 0.5 1.0 1.5 2.0 6 200 400 600 800 1000 1200 1400 1600 m/z ETD : Good fragment pattern ! ∆m = - 35 → Neutral Loss of 105 Da CID : Almost no b- and y-ions ! ? The first example deals with the identification of phosphorylation sites from different caseins. For this purpose the digested protein mixture was analyzed by alternating CID-ETD. The proteins and the phosphorylation sites were identified via Mascot database search. During the result evaluation it became obvious that several good quality ETD spectra remained unassigned. One of these spectra is shown here. The corresponding CID spectrum is shown as well, it contains only one fragment deriving from a neutral loss of 105 Da which differs quite a lot from the expected neutral loss of 98 corresponding to phosphoric acid. So the question is, what causes a neutral loss of 105 and prevents the ETD spectra from identification. Page 11 Page 11 11 11

What causes a Neutral Loss of 105 Da ? A neutral loss of 105 Da can occur from carbamidomethylated methionine:1) carbamidomethylated methionine Loss of 105 Da A neutral loss of 105 Da is already described in the literature. It occurs from carbamidomethylated methionine. The picture shows the formation of the neutral loss fragment from the carbamidomethylated methionine during the CID process. The carbamidomethylation of methionine is a sample preparation artefact. It can be formed as side product during cysteine alkylation. Carbamidomethylation of methionine is a sample preparation artefact. It can be formed as side product during cysteine alkylation. 1) Krüger et al., Rapid Commun. Mass Spectrom. 2005; 19: 1709-1716. Page 12 Page 12 12 12

NcamMAINPpSKENLCSTCK & TVDcamMEpSTEVFTKK Mascot Database Search Results for α-S2-Casein Comparison of search results without and with modification Carbamidomethyl (M) without with modification Carbamidomethyl (M) The additional modification Carbamidomethyl (M) can be included in the Mascot search parameter. Here, the search results for α-s2-casein with different search parameters are compared. The second search result was obtained considering also methionine carbamidomethylation, the first one without. Considering this modification results in the identification of two more phosphopeptides based on their ETD spectra. ► With the knowledge of camMet as sample preparation artefact, two additional phosphopeptides are identified via ETD NcamMAINPpSKENLCSTCK & TVDcamMEpSTEVFTKK Page 13 Page 13 13 13

ETD Spectrum of TVDcamMEpSTEVFTKK ETD of 551.2 (3+), tR = 11.8 min M* S* S* ... Annotation clearly confirms the presence of phosphorylated serine as well as the carbamidomethylated Methione The annotated ETD spectrum of one of the two phosphopeptides clearly shows the presence of both labile modifications: the serine phosphorylation as well as the methionine carbamidomethylation. The CID spectrum did not give an identification due to the absence of any fragments from the peptide backbone. ► A single ETD spectrum allows for the identification of phosphorylation sites also in the presence of other labile modifications. Page 14 Page 14 14 14

Strategy for glycopeptide analysis CID autoMS/MS analysis of the digested glycoprotein in enhanced resolution mode Identification of the glycopeptides: check for the presence of typical CID marker ions: - HexNAc: m/z 204 - HexNAcHex: m/z 366 - NeuAc: m/z 292, 274, 256 - HexHexNAcNeuAc: m/z 657 only for O-glycans: check for neutral loss chromatograms, e.g. for hexose (54, 81, 162), HexNAc (101.5, 203), NeuAc (145.5, 291) annotation of the sugar distances in order to determine the glycan residue ETD experiment, either in autoMSn mode with or w/o inclusion list or in manual MS/MS mode to obtain best data quality. Define the glycan moiety as modification in BioTools and match the ETD spectrum with the modified known sequence. Page 15 Page 15

Glycopeptide analysis using CID Fragments come almost exclusively from the cleavage of glycan moiety IgG3 tryptic digest glycopeptide MW 2602 Da 366.1 528.2 690.3 893.3 944.9 1025.6 1046.5 1098.9 1157.6 1200.2 1229.0 1360.7 1470.7 1506.7 1563.7 1709.8 1887.8 2400.0 400 800 1200 1600 2000 2400 pep * m/z N-acetylglucosamine galactose mannose fucose sialic acid pep Page 16 Page 16 16

Side chain cleavage of N-glyc Asn Glycopeptide analysis using ETD Fragments arise from the cleavage of peptide backbone IgG3 tryptic digest NH C=O CH3 Asn 2603.2 2054.9 2201.9 2330.0 2403.1 2458.0 2560.1 1200 1600 2000 2400 495.2 516.3 687.5 708.4 927.4 1041.4 1099.4 400 800 408.2 z3. z4. z9. 2587.1 z8. z7. z6. z5. [M+2H]2+ 1301.6 x 5 Side chain cleavage of N-glyc Asn m/z Glu-Gln-Gln-Phe-Asn-Ser-Thr-Phe-Arg z4 z5 z6 z7 z8 z9 z3 Page 17 Page 17 17

ETD CID Glycopeptide analysis using CID and ETD EQQFNSTFR CID and ETD provide complementary information for glycopeptide identification Peptide Sequence Glycan moiety Page 18 Page 18

multiply charged fragment ions up to z=4 are identified ETD analysis of large peptides galanin-like peptid (GALP) MWmono = 6200.3 Da multiply charged fragment ions up to z=4 are identified (Enhanced scan mode, 8100 m/z per sec) 3+ c23 800 805 810 815 m/z 4+ c31 2+ c16 c32 (z+1) 31 (z+1) 1 c 1 150 160 170 180 190 m/z 3+ (z+1) 50 2+ (z+1) 33 1696 1700 1704 m/z 0.0 0.5 1.0 1.5 2.0 5 x10 Intens. 500 1000 1500 2000 2500 m/z 2+ (z+1) 54 2780 2784 2788 m/z Page 19 Page 19 19

ETD of large peptides galanin-like peptid (GALP) MWmono = 6200.3 Da Deconvoluted spectrum 500 1000 1500 2000 2500 3000 c H R G W T L N S A Y P V z+1 K E c 3 c 4 c 5 c 6 c 7 c 8 c 9 c 10 c 11 c 12 c 13 c 14 c 15 z+1 15 c 16 c 17 c 19 z+1 19 c 20 c 21 z+1 21 c 22 z+1 22 c 23 z+1 23 z+1 24 c 25 z+1 25 c 26 z+1 26 c 27 z+1 27 c 28 z+1 28 z+1 29 c 30 z+1 30 c 31 z+1 31 z+1 32 3500 4000 4500 5000 5500 6000 c 32 c 33 z+1 33 c 34 z+1 34 c 35 c 36 z+1 36 c 37 z+1 37 c 38 z+1 38 c 39 c 40 z+1 41 z+1 42 c 43 z+1 43 z+1 44 c 45 z+1 46 z+1 47 c 48 z+1 48 z+1 49 c 50 z+1 50 z+1 51 z+1 52 z+1 53 z+1 54 z+1 56 c 57 z+1 57 z+1 58 z+1 59 c 60 m/z Page 20 Page 20 20

detailed characterization: preparation artefacts Use of ETD for detailed Protein Characterization protein ID detailed characterization: PTM protein termini ETD-PTR top-down analysis for the determination of N- & C-termini of intact proteins mixed modifications Protein ID is the first step in proteomics but in order to address the relevant topics a more detailed protein characterization is required. PTM analysis is an important subject because the biological activity of proteins is often determined by post-translational modifications, e.g. phosphorylation, glycosylation and so on. In the context of detailed protein characterization one needs to be able to handle multiple modifications as well as unexpected modifications. These modifications can have a natural origin or can be also preparation artefacts. For the quality control of recombinant proteins the determination of N- and C-terminus are often of great importance. Classical CID bottom-up approaches mostly fail to answer these questions whereas ETD and ETD/PTR are the perfect tools for these applications. preparation artefacts Page 21 Page 21 21

Precusor Isolation [M+12H]12+ ETD & PTR for large peptides / small proteins Ubiquitin, bovine (8559.6 Da) 500 1000 1500 2000 2500 m/z 12+ 13+ 11+ 10+ 9+ MS 500 1000 1500 2000 2500 m/z Precusor Isolation [M+12H]12+ 12+ 500 1000 1500 2000 2500 m/z 12+ 11+ 10+ 9+ ETD Page 22 Page 22

ETD & PTR for large peptides / small proteins Ubiquitin, bovine (MW = 8559.6 Da) 500 1000 1500 2000 2500 m/z ETD - PTR fragment charge states ≤ 6+ Maximum Resolution Mode PTR Proton Transfer Reaction 500 1000 1500 2000 2500 m/z 12+ 11+ 10+ 9+ ETD fragment charge states ≤ 12+ Page 23 Page 23

+ + - - 12+ Principle of ETD-PTR Top Down Analysis ETD ► Production of highly charged fragment ions from intact proteins + n+ Electron Transfer 12+ - multiply charged fragment ions n =11, 10, 9, 8, ... fragment ions with reduced charge states m = 6, 5, 4, 3, 2, 1 + Proton Transfer - n+ m+ PTR ► Charge reduction using Proton Transfer Reaction Only few fragment ion are isotopically resolved as the major amount of the fragment ions are highly charged Page 24 Page 24 24

Bruker PTR reagent from fluoranthene PTR-reagents O - Benzoate anion (Hunt, Coon et al.)  need two separate reagent reservoirs for ETD and PTR Perfluoro-1,3-dimethylcyclohexane = PDCH (McLuckey et al.) C - H Bruker PTR reagent from fluoranthene - • + H C16H10• - C16H11- Page 25 Page 25

Ubiquitin, bovine (MW = 8559.6 Da) ETD & PTR for large peptides / small proteins Deconvoluted spectrum Ubiquitin, bovine (MW = 8559.6 Da) Applications: e.g. QC of recombinant proteins, isolated proteins e.g. from cell lysates Advantages: no 1/3 cut-off, PTMs visible, good sequence coverage, N/C-termini included! Limitations: slow for LC separations, off-line techniques may be required (direct infusion, off-line nanospray, e.g. NanomateTM) Page 26 Page 26