Unambiguous Identification of Serine and ... - ACS Publications

Subscriber access provided by University of Newcastle, Australia

Article

Unambiguous identification of serine and threonine pyrophosphorylation using neutral-loss-triggered EThcD mass spectrometry Martin Penkert, Lisa M Yates, Michael Schuemann, David H. Perlman, Dorothea Fiedler, and Eberhard Krause Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05095 • Publication Date (Web): 20 Feb 2017 Downloaded from http://pubs.acs.org on February 25, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Analytical Chemistry

Unambiguous identification of serine and threonine pyrophosphorylation using neutral-losstriggered EThcD mass spectrometry

Martin Penkerta,b, Lisa M. Yatesc, Michael Schümanna,

David Perlmand, Dorothea Fiedlera,b,c,

Eberhard Krause*a a

Leibniz Institut für Molekulare Pharmakologie (FMP), Robert-Roessle Str. 10, 13125 Berlin,

Germany b

Humboldt Universität zu Berlin, Department of Chemistry, Brook-Taylor-Straße 2, 12489 Berlin,

Germany c

Princeton University, Department of Chemistry, Frick Chemistry Building, Washington Road,

Princeton, NJ 08544, USA d

Princeton University, Department of Molecular Biology, 119 Lewis Thomas Laboratory,

Washington Road, Princeton, NJ 08544, USA

Abstract Tandem mass spectrometry (MS/MS) has emerged as the core technology for identification of posttranslational modifications (PTMs). Here, we report the mass spectrometry analysis of serine and threonine pyrophosphorylation, a protein modification that has eluded detection by conventional MS/MS methods. Analysis of a set of synthesized, site-specifically modified peptides by different fragmentation techniques shows that pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178 and 196 Da, which enables the distinction between isobaric pyro- and diphosphorylated peptides. In addition, electron-transfer dissociation combined with higher energy collision dissociation (EThcD) provides exceptional data-rich MS/MS spectra for direct and unambiguous pyrophosphosite assignment. Remarkably, sufficient fragmentation of doubly charged precursors could be achieved by electron-transfer dissociation (ETD) with increased supplemental activation, without losing the labile modification. Exploiting the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation (CID), a data dependent neutralloss-triggered EThcD acquisition method was developed. This strategy enables reliable pyrophosphopeptide identification in complex samples, without compromising speed and sensitivity.

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION Covalent post-translational modifications (PTMs) modulate the structure and function of proteins. Among the most common PTMs, protein phosphorylation regulates almost all areas of cell biology, including cell signaling, regulation of enzyme activities, apoptosis and protein-protein interactions.1-4 The fundamental importance of protein phosphorylation has driven the development of specific enrichment strategies, chromatographic separation techniques, quantification methods, bioinformatic tools, and sensitive mass spectrometric methods.5-13 The reliable assignment of phosphorylation sites by tandem mass spectrometry (MS/MS) constitutes one of the most challenging tasks in MS-based phosphoproteomics.14 Owing to its sensitivity and speed, collision-induced dissociation (CID) is the most frequently used fragmentation technique for the analysis of phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) in peptides. However, the labile nature of the phosphoryl group, in particular of pSer and pThr can complicate phosphoproteomic analysis due to a dominant neutral loss of phosphoric acid, low peptide backbone fragmentation, and possible gas-phase rearrangements.14,15 To circumvent these problems, alternative strategies, including neutral-loss-triggered MS/MS/MS (MS3)16, multistage activation (MSA)17 and beam-type higher-energy collisional dissociation (HCD)18, have been applied. In addition, radical-driven fragmentation techniques, such as electron-capture dissociation (ECD) and electron-transfer dissociation (ETD) can now provide more reliable phosphosite localization.19-21 ETD is based on the transfer of an electron from a radical anion to a multiply protonated peptide ion, inducing specific N-Cα cleavages of the peptide backbone. Electron-transfer dissociation permits improved sequence coverage without the loss of labile PTMs.22 Recently, a fragmentation scheme termed EThcD was reported, which combines electron-transfer and higher-energy collision dissociation.23,24 With ETD, the more labile phosphorylation events could be analyzed, including phosphoarginine, phosphocysteine and phospholysine,.25-29 Protein pyrophosphorylation is a recently discovered, labile modification that has eluded detection by conventional MS methods because of the instability under acidic conditions.30 Embedded within polyacidic stretches, this modification occurs on phosphorylated serine residues and is mediated by the inositol pyrophosphate messengers (PP-InsPs).31-33 While a broad range of phenotypes have been linked to PP-InsPs - including insulin secretion, ribosome biogenesis in yeast, cancer cell migration and blood clotting in mammals34-38 - the role of protein pyrophosphorylation in regulating these diverse processes has not been elucidated. In fact, protein pyrophosphorylation remains a controversial topic of debate,39-41 because the characterization of protein pyrophosphorylation has exclusively relied on in vitro labeling strategies. While these in vitro studies have led to the identification of a handful of yeast and mammalian pyrophosphorylation substrates,32,42,43 direct molecular evidence for the presence of pyrophosphorylated proteins in vivo has not been presented to date. In order to understand the biological relevance and the in vivo signaling proberties of this PTM, a chemical approach for the synthesis44 and an affinity reagent for enrichment of pyrophosphorylated peptides have been


Page 2 of 19

Page 3 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


developed.45 However, a

suitable tandem mass spectrometric method for unambiguous

pyrophosphosite assignment has been lacking.41 In the present study, we synthesized a set of site-specifically pyrophosphorylated peptides based on the peptide sequences of known and putative pyrophosphorylation substrates. We analyzed these peptides, along with the corresponding isobaric diphosphorylated peptides, and studied the fragmentation behavior of doubly and triply charged precursor ions using CID, HCD, and EThcD. We show that pyrophosphopeptides can be distinguished from isobaric diphosphorylated peptides by their characteristic neutral loss pattern generated during CID. Finally, we developed a data dependent neutral-loss-triggered EThcD MS/MS (DDNL) approach, which has the potential to characterize in vitro targets and to identify endogenous protein pyrophosphorylation from complex samples.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EXPERIMENTAL SECTION Materials. For mass spectrometry analysis, LC/MS grade 0.1% formic acid in water and 0.1% formic acid in acetonitrile (ACN) were obtained from Biosolve (Valkenswaard, The Netherlands). For the preparation of synthetic peptides, dichloromethane (DCM, Reagent Grade), N,N-dimethylformamide (DMF, Reagent Plus®), N,N-dimethylacetamide (DMA, Reagent Plus®), N,N-diisopropylethylamine (DIPEA, Reagent Grade), and ACN (HPLC Grade) were purchased from Sigma Aldrich (Allentown, PA). Trifluoroacetic acid (TFA) and Pierce HeLa Protein Digest Standard was obtained from Thermo Fisher Scientific (Waltham, MA), and all amino acid building blocks, solid-phase resins, and coupling and activating reagents were purchased from Anaspec (Fremont, CA). All reagents were used as received unless otherwise specified.

Synthesis of pyro- and diphosphopeptides. The pyrophosphopeptides (PP-1-8) were synthesized using a modified procedure from Marmelstein, A.M. et. al.42 Generally, after phosphopeptide precursors were synthesized by traditional Fmoc-mode solid-phase peptide synthesis (SPPS),44 a suspension of phosphopeptide (4-6.00 µmol) in DMA was treated with a solution of Lithium benzyl 1H-imidazol-1-ylphosphonate (3 eq, 12-18.0 µmol) in DMA, followed by a solution of ZnCl2 (8 eq, 32-48.0 µmol) in H2O. The solvent mixture was typically 15% H2O in DMA (3-4.00 µM) for optimal reagent solubility. The resulting suspension was heated to 45 ˚C and stirred at this temperature for 1.5-2 hours to yield a clear solution. The reaction mixture was passed through a 0.22 µm syringe filter and directly purified by preparative high-performance liquid chromatography (HPLC). The combined product fractions were concentrated under vacuum to give the benzyl-protected intermediate as a white, crystalline solid. To a solution of the intermediate (57.00 µmol) in 15% H2O in DMA (3-4.00 µM) was added palladium on carbon (10%, 50% wet, 2 eq, 10-14.0 µmol) followed by triethylamine (4 eq, 20-28.0 µmol). The septum-topped vial was purged with an N2 atmosphere then purged with H2 gas. After stirring for 12-15 hours, the reaction was filtered through a 0.22 µm syringe filter and rinsed with H2O (100-200 µL). The resulting filtrate was directly subjected to analytical HPLC to isolate the pyrophosphorylated peptide product. The desired product fractions were concentrated under vacuum to give the title compounds as white solids. Yields are reported over two steps. The purity of the isolated peptides was confirmed by analytical HPLC, and the identity of the pyrophosphopeptides was confirmed by high-resolution mass spectrometry (HRMS) and 31P NMR. Diphosphorylated peptides (P2-1-6) were synthesized following a standard Fmoc SPPS protocol.44 The purity of the isolated peptides was confirmed by analytical HPLC, and the identity of the pyrophosphopeptides was confirmed by HRMS.


Page 4 of 19

Page 5 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


LC-MS fragmentation experiments of synthetic peptides For LC-MS/MS experiments, peptides were dissolved in water (5 pmol/µL) and analyzed by a reversed-phase capillary liquid chromatography system (Dionex Ultimate 3000 NCS-3500RS Nano, Thermo Scientific, San Jose, CA) connected to a Thermo Scientific Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific). LC separation was performed on an in-house packed 75 µm inner diameter PicoTip column (25 cm) packed with ReproSil-Pur C18AQ particles, 3 µm, 120 Å (Dr. Maisch, Germany). The flow rate was 200 nL/min using gradient of 2−30% B in 45 min. Mobilephase solvent A contained 0.1% formic acid in water and mobile-phase solvent B contained 0.1% formic acid in acetonitrile. Mass spectra were acquired in data dependent acquisition mode with a FT survey scan in the range from 350 to 1500 m/z at a resolution of 120,000 (full width at half-maximum, fwhm) followed by MS/MS scans (CID, HCD and EThcD) of the most intense precursor ions. Precursor ions were isolated with a mass selecting quadrupole within an isolation window of 1.6 m/z. CID MS/MS-spectra were measured in the linear iontrap, with a precursor AGC target value of 1e4 and normalized collision energies ranging from 25 to 3%. High resolved CID spectra were acquired with a resolution of 30,000. HCD MS/MS spectra were acquired with an AGC target value of 5e4 and normalized collision energy of 30%. EThcD spectra were measured using an AGC target value of 1e5. Calibrated charge dependent ETD parameters were used for the ETD process and HCD supplemental activation (SA) was enabled. SA collision energy was set to 50% for charge state 2 and 25% for charge state 3 (calculated on m/z and charge state of the precursor ion). HCD and EThcD MS/MS spectra were measured with a resolution of 15,000.

Neutral-loss-trigger EThcD and spike in experiments 100 fmol of peptide ISIDppTSDEESELSKK (PP-8) and 0,5 μg HeLa Protein Digest Standard were dissolved in5 μL water. Prior to every measured sequence of samples the trapping and analytical column was flushed with 0.1% citric acid in 1% of acetonitrile to chelate any residual metal ions. Using the same chromatographic conditions and instrumentation as described above, neutral-losstriggered EThcD MS/MS experiments were performed as follows: Mass spectra were acquired in data dependent acquisition mode with a FT survey scan in the range from 350 to 1500 m/z at a resolution of 120,000 (full width at half-maximum, fwhm). The maximum injection time for MS scans was set to 50 ms and the AGC target value to 2e5. Doubly and triply charged precursor ions were selected with precursor priority to the higher charge state. Precursor ions were isolated with a mass selecting quadrupole within an isolation window of 1.6 m/z. Dynamic exclusion was enabled with an exclusion interval of 10 or 40 s. CID MS/MS scans were performed with a normalized collision energy of 25% and an AGC target value of 5e3. MS/MS spectra were acquired in the iontrap with a maximum injection time of 50 ms. If neutral losses of 49 and 89 m/z from doubly charged precursor ions were



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

measured above a threshold of 15% and belonged to the five most abundant ions an additional EThcD spectra of the same precursor ions was acquired. If triply charged precursor ions showed neutral losses of 32.7 and 59.3 m/z during CID fragmentation, an additional EThcD scan was only performed if the observed signals exhibit relative intensities above 15% and were ranked among the 6 most intense signals. EThcD spectra were measured in the orbitrap with a resolution of 15,000 and the maximum accumulation time for an AGC target value of 1e5 was set to 1 s. Calibrated charge dependent ETD parameters were used for the ETD process and HCD supplemental activation was enabled. SA collision energy was set to 50% for charge state 2 and between 25 and 30% for charge state 3 (calculated on m/z and charge state of the precursor ion).

Data Analysis MS raw data were analyzed with Proteome Discoverer 2.1 software (Thermo Fisher Scientific, Bremen, Germany). To simplify EThcD spectra, the non-fragment filter was used with following parameters: Precursor ions and charged reduced precursors were removed within a 4 Da window and neutral losses within a 2 Da window. MS/MS spectra were searched against the Yeast proteome database using SEQUEST or Mascot (Matrix, Science, UK). Up to two missed cleavages were allowed for protease digestion with trypsin. Precursor mass tolerance and fragment mass deviation was set to 10 ppm and 20 mmu, respectively. Oxidation of methionine, phosphorylation (STY), pyrophosphorylation (S,T) and carbamidomethyl (C) were searched as variable modifications. Results were filtered with the Target Decoy PSM Validator and a Target false discovery rate (FDR) of 0.01 at peptide identification level. EThcD MS/MS spectra of pyrophosphorylated peptides were additionally, manually verified. Manual verification was performed considering potential gas-phase rearrangements to other phosphoacceptors, the degree of neutral loss of labile modifications, the sequence coverage of the peptide and the signal to noise ratio.

RESULTS AND DISCUSSION Behavior of pyrophosphorylated and diphosphorylated peptides during HCD & CID fragmentation To investigate the fragmentation behavior of pyrophosphopeptides (PP-peptides) in detail, a suite of peptides was synthesized using a recently described method.44 This set of peptide included characterized and putative in vitro pyrophosphorylation sites and was analyzed by LC-ESI-MS and subjected to CID and HCD fragmentation. The largest analytical hurdle will be to distinguish between pyrophosphopeptides (PP-peptides) and isobaric diphosphopeptides (P2-peptides), therefore the


Page 6 of 19

Page 7 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


corresponding P2-peptides, containing adjacent phosphorylation sites, were synthesized46 and analyzed in parallel (Table 1). Beam-type higher-energy collisional dissociation has shown to be a useful fragmentation method for phosphopeptide identification in large-scale phosphoproteome analysis.18 Indeed, HCD MS/MS fragment ion spectra of both di-and pyrophosphorylated peptides exhibited high fragment ion yields of b- and y-type ions, derived from the fragmentation at the amide bond of the peptide backbone, and allowing a reliable identification of the peptide sequence (Figure S1 & S2). During fragmentation of the PP-peptide, however, neutral loss of the labile pyrophosphate moiety was accompanied by a significant loss of water, making a reliable assignment of the modification site impossible. CID MS/MS spectra of P2-peptides revealed less peptide backbone fragmentation compared to HCD and two dominant neutral losses of 98 and 196 Da, which corresponded to the loss of one or two molecules of H3PO4, respectively. Pyrophosphorylated peptides, in contrast, exhibited a different fragmentation behavior. In addition to neutral losses of 98 and 196 Da, fragment ions corresponding to the loss of 178 Da were consistently observed with high abundance (Figure 1 & Figure S3). This distinct neutral loss, which we attribute to the elimination of pyrophosphoric acid (H4P2O7), was also observed during HCD fragmentation. It allows the differentiation between di- and pyrophosphorylated peptides and can function as a preliminary indicator for the presence of pyrophosphorylation.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 19

Table 1. Neutral loss patterns of di- and pyrophosphorylated peptides during CID fragmentation using 25% normalized collision energy. Relative intensities and ranking (Top N) of the signals corresponding to the neutral losses of 98, 178 and 196 Da are illustrated, dependent on the charge state of the precursor ion. The origin of the peptide sequences are proteins from S.cerevisiae unless indicated otherwise. peptide

peptide sequence (origin)

precursor ion

98 Da neutral loss

178 Da neutral loss

196 Da neutral loss

rel. intens. [%]

Top N

rel. intens. [%]

Top N

rel. intens. [%]

Top N

charge state (z) P2-1

VNEDSPSpSpSSK (Gcr1)

2

100

1

-

-

40

3

P2-2

SSEDpSpSEEEDKA (Nopp140-mammalian)

2

100

1

-

-

48

2

P2-3

SSDSpSpSDSESESK (AP3B1-mammalian)

2

28

4

-

-

40

2

P2-4

ISIDpSpSDEESELSKK (Puf6)

2

100

1

2

15

28

2

P2-5

RSHHDDEEEpSpSEKK (Rpa34)

2

100

1

8

4

46

2

P2-6

SKVEDAEYEpSpSDDEDEKLDK (Rrp5)

2

100

1

6

7

74

2

P2-2

SSEDpSpSEEEDKA (Nopp140-mammalian)

3

100

1

-

-

8

3

P2-4

ISIDpSpSDEESELSKK (Puf6)

3

100

1

-

-

44

4

P2-5

RSHHDDEEEpSpSEKK (Rpa34)

3

100

1

3

5

86

2

P2-6

SKVEDAEYEpSpSDDEDEKLDK (Rrp5)

3

100

1

-

-

78

2

PP-1

VNEDSPSppSSSK (Gcr1)

2

42

3

54

2

100

1

PP-2

SSEDppSSEEEDKA (Nopp140-mammalian)

2

34

3

100

1

48

2

PP-3

SSDSSppSDSESESK (AP3B1-mammalian)

2

42

2

64

2

100

1

PP-4

ISIDppSSDEESELSKK (Puf6)

2

100

1

40

2

22

3

PP-5

RSHHDDEEESppSEKK (Rpa34)

2

100

1

26

3

32

2

PP-6

SKVEDAEYEppSSDDEDEKLDK (Rrp5)

2

100

1

23

3

25

2

PP-7

SHHDDEEESppSEKKK (Rpa34)

2

100

1

20

2

10

3

PP-8

ISIDppTSDEESELSKK (Puf6;S→T)

2

100

1

28

3

34

2

PP-2

SSEDppSSEEEDKA (Nopp140-mammalian)

3

100

1

60

2

15

4

PP-4

ISIDppSSDEESELSKK (Puf6)

3

70

2

100

1

40

3

PP-5

RSHHDDEEESppSEKK (Rpa34)

3

100

1

40

2

28

3

PP-6

SKVEDAEYEppSSDDEDEKLDK (Rrp5)

3

36

2

100

1

25

3

PP-7

SHHDDEEESppSEKKK (Rpa34)

3

100

1

82

2

15

3

PP-8

ISIDppTSDEESELSKK (Puf6;S→T)

3

70

2

100

1

92

2

In agreement with previous phosphopeptide fragmentation experiments, the extent of neutral loss is influenced by several factors including the amino acid sequence of the peptide, the amino acid side chain carrying the phosphate group, the applied collision energy, and the charge state of the precursor ion.15,46-48 Compared to doubly charged precursor ions, the CID spectra of triply charged di- and pyrophosphorylated peptides exhibited reduced neutral losses, accompanied by an increased fragmentation along the peptide backbone (Figure 1). For example, the CID MS/MS spectrum of doubly charged peptide PP-4 was dominated by neutral losses of 98, 178 & 196 Da (Figure 1b), whereas the spectrum of the triply charged precursor ions showed significantly increased peptide backbone fragmentation (Figure 1d).


Page 9 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. The effect of the precursor ion charge state on the neutral loss patterns of di- and pyrophosphorylated peptides during low collision energy CID. MS/MS spectra of doubly charged peptides P2-4 (a) and PP-4 (b). MS/MS spectra of the triply charged peptides P2-4 (c) and PP-4 (d). The triply charge precursor ions show a higher extent of peptide backbone fragmentation than the doubly charged species.

Pyrophosphorylated peptides containing multiple carboxyl and/or hydroxyl residues showed a more pronounced loss of water, thereby decreasing the diagnostic 178 Da loss and increasing the 196 Da neutral loss. As an example, the CID MS/MS spectrum of peptide SSDSSppSDSESESK (PP-3), acquired with a common, normalized collision energy (NCE) of 35%, showed a low signal corresponding to the 178 Da neutral loss (Figure 2). To enhance the signal intensity of the diagnostic 178 Da neutral loss, we sought to decrease the extensive loss of water by using CID fragmentation with reduced collision energies. Indeed, the CID MS/MS spectra of peptide PP-3 showed that a reduction of the fragmentation energy suppresses the water loss (Figure 2). Applying the decreased collision energy, the 178 Da neutral loss was consistently found among the three most intense signals for the examined pyrophosphopeptides (Table 1).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Influence of collision energy on the intensities of the neutral losses of 98, 178 and 196 Da. CID MS/MS spectra of the doubly protonated peptide SSDSSppSDSESESK (PP-3) generated with a normalized collision energy (NCE) of 35%, 30% and 25%, showing that reduction of the collision energy decreases the neutral loss of 196 Da accompanied by an increase of the 178 Da neutral loss (indicated by the red lables).

To investigate whether the amino acid side chain carrying the pyrophosphate group affects the fragmentation behavior, we compared the CID MS/MS spectra of peptide ISIDppTSDEESELSKK (PP-8) with sequence ISIDppSSDEESELSKK (PP-4), which differ only in the amino acid on which the pyrophosphoryl group was installed (serine vs. threonine). The peptide containing a pyrophosphothreonine residue exhibited a similar neutral loss pattern of 98, 178 and 196 Da and a slightly higher extent of peptide backbone fragmentation (Figure S4), an observation that correlates with the fragmentation behavior of pSer and pThr peptides.48 The steric hindrance of the β-methyl group in the threonine side chain possibly causes this effect.15 The neutral loss of 178 Da can thus function as an initial indicator for the presence of pyrophosphorylated peptides; a confident assignment of the modification site by collision-based fragmentation techniques, however, remained elusive.

Unambiguous pyrophosphosite assignment using EThcD Fragment ion series of isobaric pyrophosphorylated and diphosphorylated peptides (with two adjacent phosphorylated side-chains) will only differ in one N-terminal and one C-terminal backbone fragment ion. Thus, for unambiguous characterization of a pyrophosphorylation site, complete sequence coverage (or at least fragment ions which cover the position of modifications) is required. To investigate whether ETD is suitable to definitively identify pyrophosphorylation sites, PP-peptides and P2-peptides were subjected to fragmentation. We decided to apply ETD with HCD supplemental activation, because it had previously been shown to provide data-richer spectra for unambiguous phosphosite assignment.24,28 In EThcD, precursor ions are first fragmented using ETD. The precursors, charged reduced precursors, and ETD fragment ions are then subjected to HCD supplemental activation (sa). As a result, EThcD MS/MS spectra containing b-, c-, y-, and z-ions are obtained, which enable gapless sequence confirmation of phosphopeptides without the elimination of the labile modification.24 As an example the EThcD spectrum of the triply charged precursors of peptide ACS Paragon Plus Environment

Page 10 of 19

Page 11 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ISIDppSSDEESELSKK (PP-4) is shown in Figure 3a, illustrating the complete fragmentation of the precursor ion with only minor neutral loss of pyrophosphoric acid or water. Importantly, complete sequence coverage was achieved, enabling reliable determination of the site of modification. To confirm that EThcD allows differentiation between di- and pyrophosphorylated peptides, the corresponding diphosphorylated peptide P2-4 was measured. Both a gapless c-ion series with the diagnostic fragment ions c5 and c6, and complete y- and z-ion series with ions y10 and z10, unambiguously identified the different nature of the isobaric modifications (Figure 3b & Figure S5).

Figure 3. (a) EThcD MS/MS spectra and cleavage pattern of triply charged precursor ion from the peptide PP-4. Pyrophosphosite-determining fragment ions c5, z11 and y11 are highlighted in red. (b) Detailed comparison of the EThcD MS/MS spectra of triply charged peptides PP-4 and P2-4, showing the different m/z of fragment ions c5, z10 and y10, confirming the position and the type of phosphorylation. Dashed lines highlight the differences between the di-and pyrophosphorylated peptides.

Measurements of triply charged isobaric peptides (PP-5-PP-8 & P2-5, P2-6) also generated EThcD MS/MS-spectra with full sequence coverage and intact pyrophosphoserine modifications (Figure S6 & S7), demonstrating the suitability of EThcD for analysis of pyrophosphorylated peptides.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EThcD fragmentation of doubly charged precursors Bottom-up phosphoproteomic approaches are commonly based on the enzymatic digestion of proteins with trypsin. Electrospray ionization of the generated peptides leads to the formation of precursor ions with charge states between 1 and 4, wherein doubly charged ions occur most frequently. Since charge density is an important factor influencing fragmentation efficiency50, ETD MS/MS spectra of doubly protonated peptides often reveal highly abundant signals of unfragmented charge-reduced precursor ions, impeding confident phosphosite assignment (Figure 4). To enhance fragmentation efficiency of the non-dissociative electron-transfer product, we performed EThcD experiments with greatly increased supplemental activation (see supplemental information experimental section). Remarkably, the spectra showed only moderate neutral loss of the labile modification and high fragment ion yields. As an example, the EThcD MS/MS spectrum of peptide PP-2 is displayed in Figure 4, exhibiting sufficient sequence coverage with fragment ions c5, y8 and z8, which clearly disclosed the location of the pyrophosphoryl group at serine 5. It is important to note that supplemental activation caused fragmentation of undissociated charge-reduced precursor ions, which was accompanied by an increasing extent of b- and y-ions. Interestingly, in contrast to conventional HCD fragmentation, EThcD-generated b- and y-fragment ions still carry the labile modification and show insignificantly loss of water.

Figure 4. Comparison of ETD and EThcD MS/MS spectra of doubly charged peptide PP-2 with 25% sa and 50% sa, exhibiting an enhancing fragmentation of precursor and charged reduced precursor ions leading to an extensive increase in fragment ion yield without a significant loss of the labile modification. Fragment ions which pinpoint the pyrophosphosite are labeled in red.

EThcD experiments of doubly charged precursor ions from acidic peptides PP-1 and PP-3 confirmed that ETD combined with high HCD supplemental activation provides exceptionally data-rich spectra with sufficient sequence coverage to unambiguously localize the pyrophosphorylation site (Figure S6 & S7). Fragment ions indicating phosphoryl transfer reactions in the gas-phase to other potential phosphorylation sites were not observed during any EThcD MS/MS-experiments.


Page 12 of 19

Page 13 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


On-the-fly EThcD mass spectrometry for analysis of complex samples Compared to CID and HCD, EThcD is limited by longer cycle times and lower sensitivity. To acquire a maximum number of different precursor ions in a data dependent acquisition (DDA) LC-MS/MS approach, long dynamic exclusion times (60 s), are commonly applied.51 Comparative analysis of isobaric PP-peptides and P2-peptide revealed that both species eluted within an interval of 30 s or less, with partial overlapping peak areas, during a standard 60 min chromatographic separation. The diphosphorylated peptides were detected first for all studied pairs of peptides (Figure S10), and the long dynamic exclusion times would prevent the detection of the pyrophosphorylated counterpart. Consequently, fast fragmentation techniques in combination with short exclusion times are required, to ensure the detection of both isobaric species. Therefore we combined the capabilities of CID and EThcD, and developed a data dependent neutral-loss-triggered EThcD method (DDNL-EThcD). The DDNL-EThcD method relies on an initial low collision energy CID scan, which serves to fragment as many precursor ions as possible with high sensitivity and speed. An additional EThcD scan of the same precursor ion is only triggered when both neutral losses (98 and 178 Da) appear in the ´CID filter scan`. Similar approaches have been applied in the field of phoshoproteomics, but those methods were restricted to the loss of phosphoric acid (98 Da), requiring stricter triggering conditions and leading to a significant number of missed phosphopeptide precursor ions.52,53 In contrast, the characteristic neutral loss pattern of pyrophosphorylated peptides substantially improves the specificity of the CID filter, which allows relaxed quantitative triggering requirements while simultaneously reducing the number of false positive-triggered EThcD experiments. Furthermore the DDNL-EThcD approach allows the application of short dynamic exclusion times (10-15 sec), which facilitates the detection of both isobaric species (PP-and P2-peptides) within one sample. Due to the shorter cycle time of the CID scan, the total number of acquired MS/MS experiments is increased compared to common data dependent EThcD approaches. Finally, this method enables us to use long accumulation times for the EThcD scan, leading to an increase in sensitivity. Based on the different fragmentation behavior of doubly and triply charged precursor ions during CID and EThcD, a specific data dependent decision tree was established (Figure 5).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 19

Figure 5. Data dependent decision tree of a neutral-loss-triggered EThcD method (DDNL) for analysis of pyrophosphorylated peptides. After a high resolution survey scan, measured in the orbitrap (OT), low collision energy CID is performed in the iontrap (IT). CID MS/MS spectra of doubly protonated peptides have to exhibit neutral losses of 98 and 178 Da above a relative intensity of 15% and have to belong to the five most intense peaks to trigger an extra EThcD scan with high supplemental activation. If the two neutral losses are observed within the 6th most abundant ions and above an intensity threshold of 15% in the CID-spectra of triply and higher charged precursor ions, an additional EThcD experiment is acquired. The chosen trigger requirements are based on the results of the low collision energy CID studies.

To demonstrate the capabilities of the DDNL-EThcD approach, a spike-in experiment was performed. We added 100 fmol of peptide ISIDppSSDEESELSKK (PP-4) to a HeLa protein digest. PP-4 constitutes a tryptic peptide from the protein Puf6 (Pumilio-homology domain family). Puf6 had been described in the literature as doubly phosphorylated at the positions S34 and S35;54 additionally it constitutes an in vitro target for protein pyrophosphorylation.43 The spike-in sample was analyzed by the nLC-ESI-DDNL-EThcD approach (Figure 6a). A CID MS/MS spectrum acquired at a retention time of 38.8 min exhibited the characteristic neutral loss pattern of a 98, 178 and 196 Da indicating the presence of a pyrophosphorylated peptide (Figure 6b and c). The neutral loss signals belonged to most abundant signals with relative intensities above 20% fulfilling the trigger requirements. The triggered EThcD MS/MS spectrum showed complete sequence coverage of the peptide and enabled an unambiguous assignment of the pyrophosphorylation site at the serine (S5) residue complex sample (Figure 6d).


within this

Page 15 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Detection of a synthetic pyrophosphopeptide in a spike-in experiment using the DDNL-EThcD approach. (a) Total ion chromatogram (TIC) of the HeLa cell lysate. (b) Extracted ion chromatogram (XIC) m/z 914.371 of the synthetic peptide PP-4. (c) CID MS/MS spectrum of peptide PP-4 acquired at a retention time of 38.8 min indicating dominant neutral losses of 98, 178 and 196 Da. (d) Triggered EThcD MS/MS spectrum of the pyrophosphorylated peptide ISIDppSSDEESELSKK showing gapless sequence coverage without loss of the labile modification. Fragment ions pinpointing the site of modification are labled in red.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CONCLUSION We developed a robust MS method for characterization of pyrophosphorylated peptides that can be utilized for the discovery of in vitro and in vivo pyrophosphorylated proteins. Applying commonly used CID and HCD fragmentation conditions to a set of synthetic pyrophosphorylated peptides lead to complete cleavage of the modified side-chain, preventing definitive identification of the modification site. However, CID measurements using reduced collision energy provided a characteristic neutral loss pattern, which could be used to discriminate between isobaric pyro- and diphosphorylated peptides. Pyrophosphorylated serine and threonine side-chains were stable under EThcD conditions, demonstrating that this method is suitable for unambiguous phosphosite localization of multiply protonated pyro- and diphosphorylated peptides. Remarkably, doubly charged pyrophosphorylated peptides can be fragmented without a significant loss of the labile modification by ETD using increased supplemental activation. Without compromising sensitivity or speed, a targeted data dependent neutral-loss-triggered EThcD MS/MS approach was established, which ensures the recognition and reliable phosphosite localization of pyrophosphorylated peptides. In combination with a pyrophosphopeptide enrichment, we envision that this strategy will become the method of choice for annotating pyrophosphorylation patterns, and dynamic changes thereof, to illuminate the cellular regulatory functions of this protein modification.

ASSOCIATED CONTENT Supporting Information LC chromatograms and MS spectra of peptides as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author [email protected]

Notes

The authors declare no competing financial interest.


Page 16 of 19

Page 17 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES (1) Hunter, T. Cell 2000, 100, 113-127. (2) Cohen, P. Trends Biochem. Sci. 2000, 25, 596-601. (3) Hunter, T. Phil. Trans. B-Biol. Sci. 2012, 367, 2513-2516. (4) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Cell 2006, 127, 635-648. (5) Dunn, J. D.; Reid, G. E.; Bruening, M. L. Mass Spectrom. Rev. 2010, 29, 29-54. (6) Villen, J.; Gygi, S. P. Nat. Prot. 2008, 3, 1630-1638. (7) Bodenmiller, B.; Mueller, L. N.; Mueller, M.; Domon, B.; Aebersold, R. Nat. Methods 2007, 4, 231-237. (8) Witze, E. S.; Old, W. M.; Resing, K. A.; Ahn, N. G. Nat. Methods 2007, 4, 798-806. (9) Zhou, H.; Ye, M.; Dong, J.; Corradini, E.; Cristobal, A.; Heck, A. J. R.; Zou, H.; Mohammed, S. Nat. Prot. 2013, 8, 461-480. (10) Yue, X. S.; Hummon, A. B. J. Proteome Res. 2013, 12, 4176-4186. (11) Tsai, C. F.; Hsu, C. C.; Hung, J. N.; Wang, Y. T.; Choong, W. K.; Zeng, M. Y.; Lin, P. Y.; Hong, R. W.; Sung, T. Y.; Chen, Y. J. Anal. Chem. 2014, 86, 685-693. (12) Monetti, M.; Nagaraj, N.; Sharma, K.; Mann, M. Nat.Methods 2011, 8, 655-U674. (13) Oslund, R. C.; Kee, J. M.; Couvillon, A. D.; Bhatia, V. N.; Perlman, D. H.; Muir, T. W. J. Am. Chem. Soc. 2014, 136, 12899. (14) Palumbo, A. M.; Reid, G. E. Anal. Chem. 2008, 80, 9735-9747. (15) Boersema, P. J.; Mohammed, S.; Heck, A. J. R. J. Mass Spectrom. 2009, 44, 861-878. (16) Beausoleil, S. A.; Jedrychowski, M.; Schwartz, D.; Elias, J. E.; Villen, J.; Li, J. X.; Cohn, M. A.; Cantley, L. C.; Gygi, S. P. Proc. Natl. Acad. Sci 2004, 101, 12130-12135. (17) Ulintz, P. J.; Yocum, A. K.; Bodenmiller, B.; Aebersold, R.; Andrews, P. C.; Nesvizhskii, A. I. J. Proteome Res. 2009, 8, 887-899. (18) Nagaraj, N.; D'Souza, R. C. J.; Cox, J.; Olsen, J. V.; Mann, M. J. Proteome Res. 2010, 9, 67866794. (19) Sweet, S. M. M.; Bailey, C. M.; Cunningham, D. L.; Heath, J. K.; Cooper, H. J. Mol. Cell. Proteomics 2009, 8, 904-912. (20) Collins, M. O.; Wright, J. C.; Jones, M.; Rayner, J. C.; Choudhary, J. S. J. Proteomics 2014, 103, 1-14. (21) Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E. P.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F., Proc. Natl. Acad. Sci 2007, 104 (7), 2193-2198. (22) Mikesh, L. M.; Ueberheide, B.; Chi, A.; Coon, J. J.; Syka, J. E. P.; Shabanowitz, J.; Hunt, D. F. Biochim. Biophys. Acta, Proteins Proteomics 2006, 1764, 1811-1822. (23) Frese, C. K.; Altelaar, A. F. M.; van den Toorn, H.; Nolting, D.; Griep-Raming, J.; Heck, A. J. R.; Mohammed, S. Anal. Chem. 2012, 84, 9668-9673. (24) Frese, C. K.; Zhou, H. J.; Taus, T.; Altelaar, A. F. M.; Mechter, K.; Heck, A. J. R.; Mohammed, S. J. Proteome Res. 2013, 12, 1520-1525. (25) Schmidt, A.; Ammerer, G.; Mechtler, K. Proteomics 2013, 13 (6), 945-954. (26) Trentini, D. B.; Suskiewicz, M. J.; Heuck, A.; Kurzbauer, R.; Deszcz, L.; Mechtler, K.; Clausen, T. Nature 2016, 539, 48. (27) Bertran-Vicente, J.; Serwa, R. A.; Schumann, M.; Schmieder, P.; Krause, E.; Hackenberger, C. P. R. J. Am. Chem. Soc. 2014, 136, 13622. (28) Bertran-Vicente, J.; Penkert, M.; Nieto-Garcia, O.; Jeckelmann, J.-M.; Schmieder, P.; Krause, E.; Hackenberger, C. P. R. Nat Commun 2016, 7. (29) Bertran-Vicente, J.; Schuemann, M.; Hackenberger, C. P. R.; Krause, E. Anal. Chem. 2015, 87, 6990. ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(30) Yates, L. M.; Fiedler, D. Chembiochem 2015, 16 (3), 415-423. (31) Saiardi, A.; Bhandari, R.; Resnick, A. C.; Snowman, A. M.; Snyder, S. H. Science 2004, 306, 2101. (32) Bhandari, R.; Saiardi, A.; Ahmadibeni, Y.; Snowman, A. M.; Resnick, A. C.; Kristiansen, T. Z.; Molina, H.; Pandey, A.; Werner, J. K.; Juluri, K. R.; Xu, Y.; Prestwich, G. D.; Parang, K.; Snyder, S. H. Proc. Natl. Acad. Sci 2007, 104, 15305. (33) Azevedo, C.; Burton, A.; Ruiz-Mateos, E.; Marsh, M.; Saiardi, A. Proc. Natl. Acad. Sci 2009, 106, 21161-21166. (34) Bhandari, R.; Juluri, K. R.; Resnick, A. C.; Snyder, S. H. Proc. Natl. Acad. Sci 2008, 105, 23492353. (35) Chakraborty, A.; Koldobskiy, M. A.; Bello, N. T.; Maxwell, M.; Potter, J. J.; Juluri, K. R.; Maag, D.; Kim, S.; Huang, A. S.; Dailey, M. J.; Saleh, M.; Snowman, A. M.; Moran, T. H.; Mezey, E.; Snyder, S. H. Cell 2010, 143, 897-910. (36) Illies, C.; Gromada, J.; Fiume, R.; Leibiger, B.; Yu, J.; Juhl, K.; Yang, S. N.; Barma, D. K.; Falck, J. R.; Saiardi, A.; Barker, C. J.; Berggren, P. O. Science 2007, 318, 1299-1302. (37) Rao, F.; Xu, J.; Khan, A. B.; Gadalla, M. M.; Cha, J. Y.; Xu, R. S.; Tyagi, R.; Dang, Y. J.; Chakraborty, A.; Snyder, S. H. Proc. Natl. Acad. Sci 2014, 111, 16005. (38) Ghosh, S.; Shukla, D.; Suman, K.; Lakshmi, B. J.; Manorama, R.; Kumar, S.; Bhandari, R., Blood 2013, 122 (8), 1478-86. (39) Shears, S. Proc. Natl. Acad. Sci 2010, 107, E17. (40) Majerus, P. W. Sci. Signal. 2007, 2007, pe72. (41) Saiardi, A. Biochemical Journal 2016, 473, 3765. (42) Szijgyarto, Z.; Garedew, A.;Azevedo,C.; Saiardi, A. Science 2011, 334, 802-805. (43) Wu, M.; Chong, L. S.; Perlman, D. H.; Resnick, A. C.; Fiedler, D. Proc. Natl. Acad. Sci 2016, 113, E6757. (44) Marmelstein, A. M.; Yates, L. M.; Conway, J. H.; Fiedler, D. J. Am. Chem. Soc. 2014, 136, 108111. (45) Conway, J. H.; Fiedler, D. Angew. Chem. Int. Ed. 2015, 54, 3941-3945. (46) Attard, T. J.; O'Brien-Simpson, N.; Reynolds, E. C. Int. J. Pept. Res.Ther. 2007, 13, 447-468. (47) Tholey, A.; Reed, J.; Lehmann, W. D. J. Mass Spectrom. 1999, 34, 117-123. (48) Palumbo, A. M.; Tepe, J. J.; Reid, G. E. J. Proteome Res. 2008, 7, 771-779. (49) Hoffman, M. D.; Sniatynski, M. J.; Rogalski, J. C.; Le Blanc, J. C. Y.; Kast, J. J. Am. Soc. Mass Spectrom. 2006, 17, 307-317. (50) Swaney, D. L.; McAlister, G. C.; Wirtala, M.; Schwartz, J. C.; Syka, J. E. P.; Coon, J. J. Anal. Chem. 2007, 79, 477-485. (51) Mommen, G. P. M.; Frese, C. K.; Meiring, H. D.; van Gaans-van den Brink, J. V.; de Jong, A.; van Els, C.; Heck, A. J. R. Proc. Natl. Acad. Sci 2014, 111, 4507. (52) Sweet, S. M. M.; Creese, A. J.; Cooper, H. J. Anal. Chem. 2006, 78, 7563-7569. (53) Sweet, S. M. M.; Bailey, C. M.; Cunningham, D. L.; Heath, J. K.; Cooper, H. J. Mol. Cell. Proteomics 2009, 8, 904-912. (54) Albuquerque, C. P.; Smolka, M. B.; Payne, S. H.; Bafna, V.; Eng, J.; Zhou, H. Mol. Cell. Proteomics 2008, 7, 1389.


Page 18 of 19

Page 19 of 19

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Detection of a synthetic pyrophosphopeptide in a spike-in experiment using the DDNL-EThcD approach. (a) Total ion chromatogram (TIC) of the HeLa cell lysate. (b) Extracted ion chromatogram (XIC) m/z 914.371 of the synthetic peptide PP-4. (c) CID MS/MS spectrum of peptide PP-4 acquired at a retention time of 38.8 min indicating dominant neutral losses of 98, 178 and 196 Da. (d) Triggered EThcD MS/MS spectrum of the pyrophosphorylated peptide ISIDppSSDEESELSKK showing gapless sequence coverage without loss of the labile modification. Fragment ions pinpointing the site of modification are labled in red. 726x590mm (96 x 96 DPI)


Unambiguous Identification of Serine and ... - ACS Publications

Recommend Documents