Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the

Page 1 of 26

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

Journal of Proteome Research

Optimization of LTQ-Orbitrap mass spectrometer parameters for the identification of ADP-ribosylation sites

Florian Rosenthal†,§,⊥, Paolo Nanni‡,⊥, Simon Barkow-Oesterreicher‡ and Michael O. Hottiger†,*

†

Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, CH-8057

Zurich, Switzerland ‡

Functional Genomics Center Zurich, University of Zurich, CH-8057 Zurich, Switzerland

§

Life Science Zurich Graduate School, University of Zurich, CH-8057 Zurich, Switzerland

Corresponding Author *

M.O.H. E-mail: [email protected]. Tel: +41 44 635 54 74. Fax: +41 44 635 54 68.

KEYWORDS ADP-ribosylation, histone, PARP, LTQ-Orbitrap, mass spectrometry, electron-transfer dissociation, ETD, higher energy collisional dissociation, HCD

ACS Paragon Plus Environment

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

Page 2 of 26

ABSTRACT ADP-ribosylation of proteins alters their function or provides a scaffold for the recruitment of other proteins, thereby regulating several important cellular processes. Mono- or poly-ADPribosylation is catalyzed by different ADP-ribosyltransferases (ARTs) that have different subcellular localizations and modify different amino acid acceptor sites. However, our knowledge on ADP-ribosylated proteins and their acceptor amino acids is still limited due to the lack of suitable mass spectrometry (MS) tools. Here, we describe an MS approach for the detection of ADP-ribosylated peptides and identification of the ADP-ribose acceptor sites, combining higher-energy collisional dissociation (HCD) and electron transfer dissociation (ETD) on an LTQ-Orbitrap mass spectrometer. The presence of diagnostic ions of ADP-ribose in the HCD spectra allowed us to detect putative ADP-ribosylated peptides to target in a second LC-MS/MS analysis. The combination of HCD with ETD fragmentation gave a more comprehensive coverage of ADPribosylation sites than with HCD alone. We successfully identified different ADP-ribose acceptor sites on several in vitro modified proteins. The combination of optimized HCD and ETD methods may thus be applied to complex samples, allowing comprehensive identification of ADP-ribosylation acceptor sites.


2

Page 3 of 26

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 ADP-ribosylation is an ancient post-translational protein modification (PTM) catalyzed by ADP-ribosyltransferases (ARTs)1. ARTs use nicotinamide adenine dinucleotide (NAD+) as a substrate to transfer the ADP-ribose moiety onto specific acceptor sites of target proteins or existing ADP-ribose units, thereby generating mono- or poly-ADP-ribosylated proteins1. The acceptor sites for mono-ADP-ribose are side chains of certain amino acid residues. The following amino acids have been reported to be covalent acceptor sites for ADP-ribose in eukaryotic cells: Lys, Arg, Gln, Cys, Asp and Glu, diphthamide, phospho-Ser and Asn2-6. However, these sites have primarily been identified by cumbersome chemical or mutational analyses that do not allow a comprehensive analysis of all proteins. Based on the sequence similarity to the bacterial diphtheria toxin and cholera toxin, ARTs are subdivided into ARTDs and ARTCs2, respectively. The ARTD subfamily comprises the intracellular ADP-ribosyltransferases, which are implicated in many regulatory processes and linked to various diseases and pathological conditions7-9. In cells, the ADP-ribose modification is highly transient because of the hydrolytic action of enzymes that degrade poly-ADP-ribose (PAR) to mono-ADP-ribose (MAR), as well as enzymes that remove MAR from proteins10-12. ADP-ribosylation is considered to be an element of the histone code and co-regulates gene transcription, protein function, differentiation processes, stress responses, as well as the onset and progression of diseases10, 13-16. Although research on ADP-ribosylation has a long history, the inherent technological difficulties for analyzing this complex PTM are a major reason for the large gaps in understanding of its cellular and molecular functions. Among the challenges, ADP-ribosylation can occur during sample preparation, because ARTD1 (PARP1), the main intracellular ARTD family member, is activated by fragmented DNA. On


3


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 4 of 26

the other hand, the modification is easily lost during extraction or sample preparation. The heterogeneity of the PAR chain further complicates analysis due to the unpredictable mass shift of a complex and potentially branched polymer. This problem can be addressed by enzymatically degrading PAR to MAR with poly (ADP-ribose) glycohydrolase (PARG) prior to analysis10. Another limiting factor for the functional analysis of ADP-ribosylation is the lack of suitable methods to identify and quantify the actual amino acid acceptor sites on ADP-ribosylated proteins. Without such tools it is impossible to link physiological and cellular readouts to the modification status of a specific amino acid and the activity of a certain ARTD. Liquid chromatography-mass spectrometry strategies (LC-MS/MS) are frequently used to identify specific PTMs and the most commonly employed fragmentation method is collisioninduced dissociation (CID). However, labile PTMs such as ADP-ribosylation are unstable during CID fragmentation. Indeed, a typical CID MS/MS spectrum of an ADP-ribosylated peptide is dominated by fragment ions that originate from the ADP-ribose backbone, but lacks sufficient peptide backbone fragment ions to identify the amino acid sequence17, 18. In particular,

fragment

ions

corresponding

to

adenine,

adenosine–H2O,

adenosine

monophosphate (AMP), adenosine diphosphate (ADP) and ADP-ribose are commonly observed. Recently, precursor ion scanning-triggered MS/MS strategies that monitor CID specific marker ions of ADP-ribose moiety have been proposed, allowing the selective detection of ADP-ribosylated peptides in relatively low complex protein digests19, 20. In contrast to CID, the ADP-ribose modification is maintained on the peptide during electron transfer dissociation (ETD) or electron capture dissociation (ECD), allowing the identification of the peptide sequence and the localization of the modification site. For this


4

Page 5 of 26

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


reason, these fragmentation techniques have been considered to be the best choice for the identification of ADP-ribosylation sites17, 21. Hengel et al. proposed a marker ion approach that circumvents the difficulties in peptide identification and takes advantage of the altered fragmentation pattern of ADP-ribosylated peptides21. In a first LC-MS/MS run, putative ADP-ribosylated peptides are detected (based on the presence of marker ions in the CID spectra), which are then targeted for ETD fragmentation and subsequent peptide identification in a second LC-MS/MS analysis17, 21. Although this strategy requires twice as much sample and instrument time and could be affected by the loss of low mass ions during ion trap CID fragmentation (with consequent loss of marker ion information), it allows for better characterization of ADP-ribosylated peptides. Surprisingly, the use of higher-energy collision dissociation (HCD) has only very rarely been applied to ADP-ribosylated proteins, although two recent studies have led to the identification of hundreds of ADP-ribosylated peptides22,23. In both the studies a Q-Exactive MS instrument (Thermo Scientific) was used, which allows only HCD fragmentation as dissociation technique. Since a comparison between HCD and ETD has not been performed before, we set out to systematically evaluate the use of HCD and ETD fragmentations on an ETD-enabled hybrid LTQ-Orbitrap Velos system for the identification of ADP-ribosylated peptides and their modification sites.


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 6 of 26

EXPERIMENTAL SECTION In vitro modification of standard peptide and histone mix The peptide biotin-KAARKSAPATGGVKKPHRYR (H3) or a mixture containing the four core histones as well as the H1 linker histone as full-length proteins were ADP-ribosylated as described earlier10. Briefly, 1 µg of peptide or 3 µg of histone mix were incubated with 10 pmol GST-ARTD10 (818-1025) for 15 min at 30°C in reaction buffer (50 mM Tris-HCL pH 8.0, 4 mM MgCl2, 250 µM DTT) in the presence of 160 µM NAD+. The reactions were stopped by the addition of PJ34 (ARTD inhibitor) and further processed or frozen until desalting. Nano-liquid chromatography-tandem mass spectrometry Mass spectrometry analysis was performed on an LTQ-Orbitrap Velos ETD mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to an Eksigent-NanoHPLC system (Eksigent Technologies, Dublin (CA), USA). Solvent composition at the two channels was 0.1% formic acid for channel A and 0.1% formic acid, 99.9% acetonitrile for channel B. Peptides were loaded on a self-made tip column (75 µm × 150 mm) packed with reverse phase C18 material (ReproSil-Pur 120 C18-AQ, 1.9 µm, Dr. Maisch GmbH)) and eluted at a flow rate of 300 nl per min by a gradient from 2 to 35% of B in 30 minutes (for standard peptides) or 60 minutes (for complex histone samples). Full-scan MS spectra (300−1700 m/z) were acquired at a resolution of 30 000 at 400 m/z, an accumulation gain control (AGC) of 1x106 and a maximum injection time of 250 ms. The AGC values for MS/MS analysis were set to 1x104 for ETD experiments with ion trap detection (IT, 100 ms injection time) and to 1x105 for HCD and ETD experiments with detection in the Orbitrap (FT, 200 ms injection time) respectively. The HCD normalized collision energy was set to 40%, enabling the stepped collision energy (width 15%, 3 steps), and detecting the ions at a resolution of 7500 at 400 m/z. In all the experiments, only 1 microscan was used for


6

Page 7 of 26

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


detection. The isolation width was set to 2 amu and 4 amu for HCD and ETD experiments, respectively. Full scans and Orbitrap MS/MS scans were acquired in profile mode, whereas ion trap mass spectra were acquired in centroid mode All the experiments were recorded in data-dependent manner from signals above a threshold of 2000. Charge state screening was enabled and singly charge states were rejected. For ETD experiments, the ETD anion target value was set to 1e5 and the activation time to 100 ms. Charge-state dependent ETD reaction times were enabled, setting a reference value of 100 ms for doubly charged peptides. A supplemental activation with 25% normalized collision energy was always enabled. For the analysis of standard peptides, precursor masses already selected three times for MS/MS were excluded for further selection for 45s. For the analysis of complex histone samples, the dynamic exclusion was set to 1 occurrence. The exclusion window was set to 10 ppm and the exclusion size was limited to a maximum of 500 entries. The following LC-MS/MS acquisition methods were performed: (a) Orbitrap full MS scan followed by top 10 datadependent Orbitrap HCD MS/MS; (b) Orbitrap full MS scan followed by top 10 datadependent ion trap ETD MS/MS, or (c) top 8 data-dependent Orbitrap ETD MS/MS; (d) Orbitrap full MS scan followed by top 10 double-play data-dependent HCD MS/MS and ion trap ETD MS/MS; (e) Orbitrap full MS scan followed by top 10 data-dependent HCD and an additional ETD ion trap event when a peak at 136.0623 (ADP-ribose fragment ion) was observed among the top 10 most abundant peaks of the HCD scan (HCD-PD1-ETD); (f) Two-stage, consisting of HCD and an additional ETD ion trap MS/MS acquisition on the top 10 precursors where the marker ions 136.0623, 250.0940 and 348.07091 were observed in a previous HCD run. A complete description of all tandem MS experiments employed in this study can be found in Table 1.


7


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 26

Database analysis and configuration of Mascot modifications MS and MS/MS spectra were converted into Mascot generic format (mgf) using Proteome Discoverer v1.4 (Thermo Fisher Scientific, Bremen, Germany). When both HCD and ETD scans were presents in the same raw files, separate mgfs for the two fragmentation techniques were created. All high-resolution HCD and ETD MS/MS spectra were deconvoluted using MS Spectrum Processor v0.924. In the ETD spectra the precursor, the charge-reduced precursor(s) and the neutral losses were removed. The mgfs were searched against the UniProtKB human database (taxonomy 9606, version 20140422), including 35787 SwissProt, 37802 TrEMBL entries, 73589 decoy hits, and 260 common contaminants. Mascot 2.4.1 (Matrix Science) was used for the identification with the following search settings: the combined HCD and Orbitrap ETD mgfs were searched with a peptide tolerance of 10 ppm and MS/MS tolerance of 0.05 Da, while the MS/MS tolerance for the ion trap ETD spectra was set to 0.6 Da. For the HCD runs, singly charged b and y ion series, immonium ions, water and ammonia loss ion series were searched. For ETD, multiply charged c, y, z, z+1, z+2 series were considered. Enzyme specificity was set to trypsin, allowing up to 4 missed cleavages. Decoy hits were used to control the false discovery rate at the peptide and protein level. The ADP-ribose variable modification was set up differently for the searches of HCD or ETD spectra. For HCD mgfs the modification was set to a mass shift of 541.0611, with scoring of the neutral losses equal to 347.0631 and 249.0862. The marker ions at m/z 428.0372, 348.0709, 250.0940, 136.0623 were ignored for scoring. Nine additional neutral losses were set as satellite (Fig. S1). For the identification of ADP-ribosylation from ETD spectra, only the mass shift of 541.0611 was used. An ADP-ribosylated peptide was considered as correctly identified when a Mascot score higher than 20 and an expectation value lower than 0.05 was obtained. To assess the location of the ADP-ribosylation sites, we used the site localization analysis provided by Mascot, which is based on the work by


8

Page 9 of 26

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


Savitski et al.25 and was developed especially for phosphorylation. This method is not optimized for ADP-ribosyl modification, due to the lack of standard peptides with known modification sites. For this reason, even if Mascot states a correctness of 95% for the site localization, this value is arbitrary and cannot be experimentally validated. Due to the lack of a better estimate, we define correctness as having a confidence of at least 95% in the Mascot site localization analysis. PTM MarkerFinder For LC-MS/MS experiments where HCD fragmentation has been used, the Mascot output .dat files have been further analyzed using PTM MarkerFinder26. Briefly, PTM MarkerFinder screens the Mascot outputs for HCD spectra containing the ADP-ribose marker ions at m/z 136.0623, 250.0940, 348.0709 and 428.0372. The spectra containing at least two marker ions and where the sum of the marker ion intensities covers at least 5 % of the total ion intensities are considered as spectra from putative ADP-ribosylated peptides. Finally, PTM MarkerFinder summarizes information about the presence and the intensity of marker ions and annotates the spectra with the corresponding peptide sequence.


9


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 10 of 26

RESULTS AND DISCUSSION Optimal HCD fragmentation of the ADP-ribosylated standard peptide using ramped CE The currently available MS/MS protocols are not optimized for the accurate and reliable detection of ADP-ribosylated peptides and their acceptor sites in complex samples. To improve MS-based detection of complex ADP-ribosylated peptides, three model samples with different complexities were prepared as described in the Experimental Section. To better comprehend the behavior of ADP-ribosylated peptides during HCD fragmentation and to detect the diagnostic ions characteristic for the ADP-ribose moiety of modified peptides, an ADP-ribosylated H3 standard peptide was first fragmented using HCD with NCE (normalized collision energy) values ranging from 23% to 60% (Fig. 1A,B and as described by Hengel et al.17). This analysis revealed that the MARylated standard peptide showed high intensity signals for the expected ADP-ribose marker ions, but low intensity signals for the peptide fragment at higher NCE values (Fig. S2). A good balance between the intensity of ADP-ribose marker ions and peptide backbone fragments was achieved by using ramped NCE (width 15%, 3 steps centered around 40%). The HCD spectra acquired with such parameters were dominated by the marker ions originating from the internal fragmentation of ADP-ribose (with the ion at 136 being the most abundant one), as well as by additional fragments corresponding to the peptide plus a residual mass from ADP-ribose fragmentation (Fig. 1C). These ADP-ribose fragments are often not caused by neutral losses and may thus exhibit charge states different to the one of the precursor. Based on these results, ramped NCE was defined as the optimal setting and used for all following analyses. Moreover, the Mascot settings for the ADP-ribose modification were improved by: (i) exclusion of the marker ion for scoring; (ii) use of m6 and m3 neutral losses for scoring; (iii) use of m1, m1+H2O,


10

Page 11 of 26

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


m1+2H2O, m3+H2O, m6+H2O, m8, m8+H2O, m8+2H2O, and m10 as satellite neutral losses (Fig. 1C, Fig. S1).

The combination of HCD and ETD fragmentation increases the number of identified ADPribosylation sites from a mixture of in vitro modified full-length histone proteins Until recently, ETD fragmentation was considered the method of choice for detecting and identifying ADP-ribosylated peptides and was therefore compared to HCD fragmentation (Table 1 / Fig. 2). In a next step, a mixture of all four core histones and linker histone H1 was ADP-ribosylated by ARTD10 (818-1025) in vitro and all the resulting peptides were analyzed by HCD (as described above) or by ETD fragmentation acquired in the ion trap (IT) or Orbitrap (FT). Since the average charge states of the identified peptides were relatively low (less than 4, Fig. S3), the acquisition of high-resolution ETD (FT) MS/MS spectra did not lead to identification of a higher number of unique ADP-ribosylated peptides (Fig. 3B). In contrast, the acquisition of the ETD spectra in the IT allowed a higher throughput compared to FT detection (due to the shorter length of the scan cycles), achieving a slightly higher number of identifications (Fig. 3). When ADP-ribosylated histone samples were analyzed by either HCD or ETD fragmentation alone, HCD resulted in nearly 2 times more ADP-ribosylated peptide spectrum matches (PSMs) than ETD (Fig. 3A). In principle, combining ETD and HCD may improve the identification of ADP-ribosylated peptides and modification sites, as it takes advantage of the information generated by both approaches. However, subjecting the sample to HCD-ETD (IT) results in increased cycle times and in a lower number of identifications and assigned spectra (Fig. 3). This strategy is particularly inefficient for complex samples, because all peptides, also the ones that are not modified, are fragmented and analyzed twice. To overcome these shortcomings, HCD was


11


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 12 of 26

followed by ETD on the same precursors only when one or more marker ions were present in the HCD spectra (product-dependent approach, HCD-PD-ETD17). While this approach has been successfully used for glycopeptide studies27-29, this is the first description, to the best of our knowledge, of it being used for the identification of ADP-ribosylated peptides using an LTQ-Orbitrap machine. The ETD (IT) was triggered upon the detection of the marker ion at m/z 136.0623 (HCD-PD1-ETD (IT)). In general, the marker ion 136.0623 proved specific enough for the triggering of ETD fragmentation, while the inclusion of 3 marker ions (136.0623, 250.0940, 348.0709 (HCD-PD3-ETD (IT)) increased the number of missed PDETD activations (data not shown). The HCD-PD1-ETD (IT) approach identified approximately the same number of unique peptides as the two single HCD and ETD (IT) runs together, indicating that all modified peptides with a clear ion pattern were already detected and identified by the two single runs (Fig. 3B). The previously suggested two-stage (“marker ion”) approach17, which has not yet been tested with HCD and ETD, is an alternative method. To evaluate this method, samples were first analyzed using HCD fragmentation. Subsequently, the Mascot output was screened for spectra containing ADP-ribose marker ions using PTM MarkerFinder26 to obtain a list of precursor ions corresponding to putative ADP-ribosylated peptides. Only these precursor ions were targeted for HCD and ETD (IT) fragmentation in a second LC-MS/MS experiment. Interestingly, when compared to the HCD-PD1-ETD (IT) approach, the two-stage HCD-ETD (IT) experiment identified almost twice as many ADP-ribosylated PSMs, but fewer unique ADP-ribosylated peptides (Fig. 3B). However, to increase the chances of acquiring good quality MS/MS spectra, we used different settings in dynamic exclusions between single HCD and marker ion-dependent runs (after 1 and 3 occurrences, as described in the Experimental Section). Despite the different repeat counts, chosen for optimal performance,


12

Page 13 of 26

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


the difference in output may indicate that the more abundant modified peptides were repeatedly selected for MS/MS and identified with a high score, thus limiting the detection of less abundant ones. To validate the potential benefits of the ETD fragmentation in these approaches, the contributions from HCD and ETD spectra to the identified peptides from the combined HCDETD (IT) runs were compared. A significant proportion of the unique ADP-ribosylated peptides was identified uniquely using ETD, suggesting that the ETD fragmentation provided additional information compared to HCD alone (Fig. 3C). Taken together, the identification and mapping of the ADP-ribosylation sites in a more complex sample was best achieved by the application of HCD-PD1-ETD (IT) or the twostage HCD-ETD (IT) approach. Both methods identified a large number of acceptor sites and recorded the additional ETD-information. However, in most cases, a single HCD run might be sufficient to capture the majority of ADP-ribosylated peptides and their acceptor sites. Two things have to be taken into consideration if complex cellular extracts are to be analyzed. As complex mixtures also contain poly-ADP-ribosylated peptides, an additional sample handling step that reduces the complexity of the PAR structure can significantly increase the number of successful identifications. Poly-ADP-ribose can be reduced to monoADP-ribose by treatment with PAR-degrading enzymes such as PARG or ARH3, thereby creating a defined mono-ADP-ribose moiety that can be reliably detected with our optimized method. In addition, ADP-ribosylation is not abundant in cellular extracts and most attempts to identify the ADP-ribosylome without prior enrichment have failed or have generated only poor results. Therefore, recent studies have employed different methods to enrich ADPribosylated peptides from complex mixtures to successfully identify ADP-ribosylated proteins22,

23, 30

. However, the unambiguous and unbiased site localization still remains a


13


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 26

technical challenge and the combination of the herein described new site identification methods, together with PARG pre-treatment and the enrichment of ADP-ribosylated peptides, is therefore a powerful tool to identify the cellular ADP-ribosylome. The different MS/MS protocols described above led to the identification of 167 ADPribosylated peptides (854 PSMs) of in vitro modified ARTD10 and histones (Table 2 and Table S1). Several sites or stretches of ADP-ribosylation were covered by overlapping peptides, underlining the robustness of this approach. Interestingly, ADP-ribosylation of histones by ARTD10 in vitro was not limited to a single type of amino acid residue; modifications were found on Glu and Asp, as well as on Lys and Arg. Representative HCD (FT) and ETD (IT) annotated spectra of ADP-ribosylated peptides modified on Glu, Asp, Lys and Arg are shown in Supplementary Fig. S4 and S5, respectively. Previously, mainly Glu and Asp residues were identified as automodification sites for ARTD10, an enzyme that due to substrate-assisted catalysis is predicted to modify acidic amino acids of selected modified target proteins in vitro31. However, our analyses imply that ARTD10 is able to modify more than one type of amino acid in vitro. Whether the identified basic acceptor amino acids are modified also by a substrate-assisted mechanism remains to be determined.

CONCLUSIONS The approach for the reliable identification of ADP-ribosylated proteins and their specific modification sites described in the present study combines methods for the preparation of ADP-ribosylated samples, data acquisition on an LTQ-Orbitrap system and data analysis (Fig. 1A, 2A). The combination of HCD and ETD fragmentations, preferably by HCD-PD1ETD (IT) or two-stage HCD-ETD (IT), enables the identification of ADP-ribosylation sites


14

Page 15 of 26

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


with high reliability and confidence of in vitro modified proteins. With these approaches, we were able to identify several ADP-ribose acceptor sites (Glu, Asp, Lys and Arg) on various different proteins with high confidence. We thus present a robust method for the exact identification of ADP-ribosylation sites using an ETD-enabled LTQ-Orbitrap mass spectrometer that can be applied to various proteins of interest and which expands the possibility to reliably identify ADP-ribose acceptor sites in complex samples such as cellular extracts.

ASSOCIATED CONTENT Supporting Information. Supplementary Fig. 1: Mascot settings used for the configuration of ADP-ribose modification.

Supplementary

Fig.

2:

HCD

spectra

of

peptide

biotin-

KAARKSAPATGGVKKPHRYR + 1 ADP-ribose. Supplementary Fig. 3: Average charge state of the identified ADP-ribosylated peptides. The average charge state was defined as the mean of all charge states. Supplementary Fig. 4: Representative HCD (FT) annotated spectra of ADP-ribosylated peptides modified on Glu, Asp, Lys and Arg. Supplementary Fig. 5: Representative ETD (IT) annotated spectra of ADP-ribosylated peptides modified on Glu, Asp, Lys and Arg. Supplementary Table 1: List of ADP-ribosylated peptides identified from in vitro modified full-length histone proteins with unambiguous site localization (>95%). Supplementary Table 2: List of ADP-ribosylated peptides identified from in vitro modified full-length histone proteins with ambiguous site localization (95%)

protein name ARTD10 ARTD10 ARTD10 Histone H2A Histone H2A Histone H2A Histone H2A Histone H2A Histone H2A Histone H2A Histone H2A.Z Histone H2B Histone H2B Histone H2B Histone H2B Histone H2B Histone H2B Histone H2B Histone H2B Histone H2B Histone H2B Histone H2B Histone H3.1 Histone H3.1 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4 Histone H4

peptide sequence LAENTGEFQEVVR RPVEQVLYHGTTAPAVPDICAHGFNR YDSAVDCICQPSIFVIFHDTQALPTHLITCEHVPR HLQLAIRNDEELNKLLGR HLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPK HLQLAIRNDEELNKLLGKVTIAQGGVLPNIQAVLLPK NDEELNKLLGK SSRAGLQFPVGR VGAGAPVYLAAVLEYLTAEILELAGNAAR VGAGAPVYLAAVLEYLTAEILELAGNAARDNKK VGATAAVYSAAILEYLTAEVLELAGNASK AMGIMNSFVNDIFER EIQTAVRLLLPGELAK HAVSEGTKAVTKYTSAK KESYSVYVYK LLLPGELAK QVHPDTGISSK QVHPDTGISSKAMGIMNSFVNDIFER SRKESYSVYVYK SRKESYSVYVYK VLKQVHPDTGISSK VLKQVHPDTGISSK EIAQDFKTDLR FQSSAVMALQEACEAYLVGLFEDTNLCAIHAKR DAVTYTEHAK DAVTYTEHAKR GVLKVFLENVIR GVLKVFLENVIRDAVTYTEHAK ISGLIYEETR ISGLIYEETRGVLK KTVTAMDVVYALKR RISGLIYEETR RISGLIYEETR TVTAMDVVYALKR VFLENVIR VFLENVIRDAVTYTEHAK VFLENVIRDAVTYTEHAK

peptide mz

charge

peptide mr

peptide score

expect value

confidence

protein accession

No. of PSM's

1016.9029 862.3815 937.0075 891.7522 912.2855 912.4857 605.2487 605.9244 1152.8828 986.23 1146.204 1142.9429 764.7054 580.5065 602.9036 747.8319 570.5568 859.3685 683.9515 683.9467 683.9728 683.969 626.255 1075.7166 559.2044 611.2402 643.306 761.6124 574.5655 706.9855 712.657 626.5988 626.5963 669.9571 765.8221 662.2984 882.7225

2+ 4+ 5+ 3+ 5+ 5+ 3+ 3+ 3+ 4+ 3+ 2+ 3+ 4+ 3+ 2+ 3+ 4+ 3+ 3+ 3+ 3+ 3+ 4+ 3+ 3+ 3+ 4+ 3+ 3+ 3+ 3+ 3+ 3+ 2+ 4+ 3+

2031.7912 3445.4968 4680.0012 2672.2348 4556.3909 4557.3922 1812.7242 1814.7515 3455.6266 3940.8911 3435.5901 2283.8712 2291.0943 2317.997 1805.6891 1493.6493 1708.6486 3433.445 2048.8328 2048.8183 2048.8965 2048.8851 1875.7432 4298.8371 1674.5914 1830.6987 1926.8961 3042.4204 1720.6746 2117.9346 2134.9491 1876.7746 1876.7669 2006.8496 1529.6296 2645.1645 2645.1458

66.92 97.51 60.11 51.42 71.72 69.86 47.71 53.39 71.3 41.88 39.94 90.12 29.15 31.77 64.02 21.55 32.61 30.91 38.07 25.27 59.24 29.68 43.36 95.04 43.94 30.27 39.04 52.91 38.89 54.14 39 55.27 56.43 37.2 27.76 35.85 74.09

0.00000099 7.2E-10 0.0000023 0.00011 0.000063 0.000049 0.00053 0.000048 0.00000025 0.00027 0.00018 8.6E-09 0.0019 0.038 0.0000054 0.039 0.0016 0.012 0.00042 0.033 0.00016 0.002 0.001 1.5E-09 0.0002 0.0096 0.023 0.0023 0.0024 0.000018 0.00022 0.000052 0.0000066 0.00032 0.0038 0.00043 0.000021

98.1% 97.6% 98.3% 95.1% 98.7% 98.6% 97.3% 100.0% 99.8% 99.7% 95.1% 97.7% 99.7% 95.3% 98.3% 99.0% 100.0% 98.3% 95.6% 96.8% 100.0% 99.2% 97.6% 96.1% 99.9% 97.0% 97.6% 99.8% 97.1% 95.2% 99.2% 95.7% 95.3% 100.0% 100.0% 95.9% 98.8%

tr|E9PK67|E9PK67_HUMAN tr|E9PK67|E9PK67_HUMAN tr|E9PK67|E9PK67_HUMAN sp|P04908|H2A1B_HUMAN sp|Q6FI13|H2A2A_HUMAN sp|Q6FI13|H2A2A_HUMAN sp|P0C0S8|H2A1_HUMAN sp|Q6FI13|H2A2A_HUMAN sp|P04908|H2A1B_HUMAN sp|P04908|H2A1B_HUMAN sp|P0C0S5|H2AZ_HUMAN sp|P57053|H2BFS_HUMAN sp|O60814|H2B1K_HUMAN sp|O60814|H2B1K_HUMAN sp|P57053|H2BFS_HUMAN tr|B4DR52|B4DR52_HUMAN sp|P57053|H2BFS_HUMAN sp|Q8N257|H2B3B_HUMAN sp|O60814|H2B1K_HUMAN tr|B4DR52|B4DR52_HUMAN sp|P57053|H2BFS_HUMAN sp|Q8N257|H2B3B_HUMAN sp|P68431|H31_HUMAN sp|P68431|H31_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN sp|P62805|H4_HUMAN

4 4 1 1 4 2 1 2 4 5 1 3 4 1 1 4 6 4 1 1 5 7 1 1 10 4 1 1 1 1 1 1 1 6 23 1 2

ADP-ribosylation sites are highlighted in red. All ADP-ribosylated peptides with a Mascot localization score >95% are listed. PSMs: peptide spectrum matches.



Figure 1

Rosenthal et al.

A.

OpƟmizaƟon of HCD parameters

B.

HCD

int

ARTD10

RT

m/z

LC- MS

MS/MS

m6 m8

m3

OH

O P O O OH

O p3

OH

O

O

p8 p10

p5

OH

OH

Peak list extracƟon

Mascot DB search

/ĚĞŶƟĮĐĂƟŽŶŽĨ WͲƌŝďŽƐǇůĂƟŽŶ

PTM MarkerFinder

OH

O

P

OpƟmizaƟon of Mascot modiĮcaƟons

int

H3 pepƟde

m1+ : 136.06 m3+ : 250.09 m6+ : 348.05 m8+ : 428.02 m10+: 542.07

m1

N N

N N

(adenine+) (adenosine -H2O+) (adenosine-mono phosphate+ (AMP+)) (adenosine-di phosphate+ (ADP+)) (ADP-ribose+)

NH2

C.

100

590.2837 z=5

25

30 136.0607 z=1

348.0678 z=1 m

0

250.0917 z=1

200

400

0

675.3312 z=4 939.5150 802.7794 z=1 z=3

+

8

600

800

586.6831 z=5

10

m3+

10 5

20

650.8375 z=4

m6+

20 15

1000 m/z

646.3342 z=4 641.8329 z=4

655.5885 z=4

675.3312 z=4

-H2O

[M+4H]4+-m1

[M+4H]4+-m3

[M+4H]4+-m6 650.8375 z=4

621.5906 z=4

699.3364 z=4

580 590 600 610 620 630 640 650 660 670 680 690 700 710 m/z 1681.2023 z=?

1200

-H2O

[M+4H]4+-m8

602.3379 z=4

-H2O

40

-H2O

30

m1+

-H2O

35

-H2O

40

50

-H2O

45

60

-H2O

95

70

[M+4H]4+-m10

100

80

-H2O

590.2837 z=5

-H2O

FTMS + p NSI d Full ms2 [email protected] [100.00-2000.00]

-H2O

Relative Abundance

90

Relative Abundance

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 24 of 26

1400

1600

1800

1951.7073 z=?

2000

1 ACS Paragon Plus Environment

Page 25 of 26

Figure 2

Rosenthal et al.

int

HCD ADP-ribose marker ion

m/z

int

ETD (IT)

m/z

int

ETD (FT)

MS/MS

m/z

1 marker ion m/z

PTM MarkerFinder

HCD RT

m/z

+

ETD (IT) int

HCD

/ĚĞŶƟĮĐĂƟŽŶŽĨ WͲƌŝďŽƐǇůĂƟŽŶ

ETD (IT)

int

m/z

m/z

Product Dependent

int

m/z

HCD

ETD (IT)

int

LC- MS

+

Mascot DB search

int

HCD int

RT

Peak list extracƟon

m/z

int

ARTD10

int

histone mixture

int

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


m/z



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 26 of 26

Figure 3

Rosenthal et al.


Optimization of LTQ-Orbitrap Mass Spectrometer Parameters for the

Recommend Documents