Tyrosine sulfation in a Gram-negative bacterium

Han, Sang-Wook; Lee, Sang Won; Bahar, Ofir; Schwessinger, Benjamin; Robinson, Michelle; Shaw, Jared; Madsen, James A.; Brodbelt, Jennifer S.; Ronald, Pamela C.

doi:10.1038/ncomms2157

Cited by 64 publications

(72 citation statements)

References 28 publications

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“…This trend was consistent for 1-, 2-, and 3- precursor ions. These results align well with previous UVPD studies that favored the use of minimal laser pulses and energy for fragmentation of deprotonated peptides [38–40,42]. When the abundances of SO 3 neutral loss ions from precursor and charge-reduced precursor ions were tabulated, normalized, and averaged across peptides (Supplemental Figure 10), the results showed the same pattern that was observed for a/x sequence ions, with the SO 3 neutral loss ions increasing in abundance as fewer laser pulses at lower energy were used for activation.…”

Section: Resultssupporting

confidence: 91%

“…Ultraviolet photodissociation (UVPD) at 193 nm is a fast activation method that has shown merits for peptide sequencing in both the positive and negative modes [34–40], including analysis of peptides decorated with acidic PTMs [38,41,42]. Upon UVPD activation, peptide anions dissociate into predominantly a and x type ions with other ion series including c, z, and y observed less frequently.…”

Section: Introductionmentioning

confidence: 99%

“…Similar promising results have recently been obtained for a sulfated peptide derived from the Ax21 protein in the gram negative bacterium Xanthomonas oryzae pv. oryzae [42]. This first successful identification of tyrosine sulfation using UVPD has prompted a more in-depth investigation of UVPD analysis of deprotonated sulfopeptides.…”

Section: Introductionmentioning

confidence: 99%

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Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

Robinson

Moore

Brodbelt

2014

J. Am. Soc. Mass Spectrom.

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Sulfation is a common post-translational modification of tyrosine residues in eukaryotes; however, detection using traditional liquid chromatography-mass spectrometry (LC-MS) methods is challenging based on poor ionization efficiency in the positive ion mode and facile neutral loss upon collisional activation. In the present study, 193 nm ultraviolet photodissociation (UVPD) is applied to sulfopeptide anions to generate diagnostic sequence ions which do not undergo appreciable neutral loss of sulfate even using higher energy photoirradiation parameters. At the same time, neutral loss of sulfate is observed from the precursor and charge reduced precursor ions, a spectral feature that is useful for differentiating tyrosine sulfation from the nominally isobaric tyrosine phosphorylation. LC-MS detection limits for UVPD analysis in the negative mode were determined to be around 100 fmol for three sulfated peptides, caerulein, cionin, and leu-enkephalin. The LC-UVPD-MS method was applied for analysis of bovine fibrinogen, and its key sulfated peptide was confidently identified.

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Section: Resultssupporting

confidence: 91%

Section: Introductionmentioning

confidence: 99%

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Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

Robinson

Moore

Brodbelt

2014

J. Am. Soc. Mass Spectrom.

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“…The analyzed spectra were the result of fifty averaged scans. The Ser5 monophosphorylated and Thr/Ser5 diphosphorylated peptides were also analyzed using dual selected ion monitoring (SIM) on an Orbitrap Elite mass spectrometer (ThermoFisher, San Jose, CA) customized to accommodate 193 nm ultraviolet photodissociation (UVPD) via an excimer laser in the HCD cell as described previously (29). Dual selected ion monitoring (SIM) scans were performed encompassing the expected mass-to-charge ratios of both the unreacted and the dephosphorylated peptides.…”

Section: Methodsmentioning

confidence: 99%

Novel Modifications on C-terminal Domain of RNA Polymerase II Can Fine-tune the Phosphatase Activity of Ssu72

et al. 2013

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The C-terminal domain of RNA polymerase II (CTD) modulates the process of transcription through sequential phosphorylation/dephosphorylation of its heptide repeats through which it recruits various transcription regulators. Ssu72 is the first characterized cis-specific CTD phosphatase that dephosphorylates Ser5 with a requirement for the adjacent Pro6 in a cis conformation. The recent discovery of Thr4 phosphorylation in the CTD calls into question whether such a modification can interfere with Ssu72 binding via the elimination of a conserved intra-molecular hydrogen bond in the CTD that is potentially essential for recognition. To test if Thr4 phosphorylation will abolish Ser5 dephosphorylation by Ssu72, we determined the kinetic and structural properties of Drosophila Ssu72-symplekin in complex with the CTD peptide with consecutive phosphorylated Thr4 and Ser5. Our mass spectrometric and kinetic data established that Ssu72 doesn’t dephosphorylate Thr4, but the existence of phosphoryl-Thr4 next to Ser5 reduces the activity of Ssu72 towards the CTD peptide by four fold. To our surprise, even though the intra-molecular hydrogen bond is eliminated due to the phosphorylation of Thr4, the CTD adopts an almost identical conformation to be recognized by Ssu72 with Ser5 phosphorylated alone or both Thr4/Ser5 phosphorylated. Our results indicate that Thr4 phosphorylation will not abolish the essential Ssu72 activity, which is needed for cell survival. Instead, the phosphatase activity of Ssu72 is fine-tuned by Thr4 phosphorylation and eventually may lead to changes in transcription. Overall, we report the first case of structural and kinetic effects of phosphorylated Thr4 on CTD modifying enzymes. Our results support a model in which a combinatorial cascade of CTD modification can modulate transcription.

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“…Photodissociation was implemented in a manner similar to that described previously (32,33). LC separations were performed using water (A) and acetonitrile (B) mobile phases containing 0.05% acetic acid (pH ϳ 4) on a Dionex Ultimate 3000 configured for pre-concentration with a loading solvent of 98% water, 2% acetonitrile, and 0.05% acetic acid.…”

Section: Methodsmentioning

confidence: 99%

High-throughput Database Search and Large-scale Negative Polarity Liquid Chromatography–Tandem Mass Spectrometry with Ultraviolet Photodissociation for Complex Proteomic Samples

Madsen

Robinson

et al. 2013

Molecular & Cellular Proteomics

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The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS 1 and MS 2 data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples, respectively, with 49 and 50 unique proteins identified in contrast to the more conventional MS/MS methods. The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public. The accuracy and specificity of the MassMatrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap. Analysis of a known mixture of three mitogen-activated kinases yielded similar sequence coverage percentages for UVPD of peptide anions versus conventional collisioninduced dissociation of peptide cations, and when these methods were combined into a single search, an increase of up to 13% sequence coverage was observed for the kinases. The ability to sequence peptide anions and cations in alternating scans in the same chromatographic run was also demonstrated. Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species. In this case, tryptic peptides from the cytosolic section of HeLa cells were analyzed by polarity switching nanoLC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing. Relative to searching using ETD alone, positive/negative polarity switching significantly improved sequence coverages across identified proteins, resulting in a 33% increase in unique peptide identifications and more than twice the number of peptide spectral matches. Molecular & Cellular Proteomics 12: 10.1074/mcp.O113.028258, 2604-2614, 2013.The advent of new high-performance tandem mas...

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Tyrosine sulfation in a Gram-negative bacterium

Cited by 64 publications

References 28 publications

Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

Direct Identification of Tyrosine Sulfation by using Ultraviolet Photodissociation Mass Spectrometry

Novel Modifications on C-terminal Domain of RNA Polymerase II Can Fine-tune the Phosphatase Activity of Ssu72

High-throughput Database Search and Large-scale Negative Polarity Liquid Chromatography–Tandem Mass Spectrometry with Ultraviolet Photodissociation for Complex Proteomic Samples

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