ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry

LeDuc, Richard D.; Taylor, Gregory K.; Kim, Yong Bin; Januszyk, Thomas E.; Bynum, Lee; Sola, Joseph V.; Garavelli, John S.; Kelleher, Neil L.

doi:10.1093/nar/gkh447

Cited by 183 publications

(188 citation statements)

References 13 publications

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“…Relative ratios of alkylated forms of p53 were calculated from peak heights [36]. The SNAP 2.0 algorithm was used for automated peak picking of top-down and middle-down fragmentation data and the resulting mass lists were searched against the relevant primary sequences using ProSight PTM [37] or Biotools (Bruker Daltonics) software. Error tolerances were 10 ppm.…”

Section: Discussionmentioning

confidence: 99%

Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

Scotcher

Clarke

Weidt

et al. 2011

J. Am. Soc. Mass Spectrom.

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The tumor suppressor p53 is a redox-regulated transcription factor involved in cell cycle arrest, apoptosis and senescence in response to multiple forms of stress, as well as many other cellular processes such as DNA repair, glycolysis, autophagy, oxidative stress and differentiation. The discovery of cysteine-targeting compounds that cause re-activation of mutant p53 and the death of tumor cells in vivo has emphasized the functional importance of p53 thiols. Using a combination of top-down and middle-down FTICR mass spectrometry, we show that of the 10 Cys residues in the core domain of wild-type p53, Cys182 and Cys277 exhibit a remarkable preference for modification by the alkylating reagent N-ethylmaleimide. The assignment of Cys182 and Cys277 as the two reactive Cys residues was confirmed by site-directed mutagenesis. Further alkylation of p53 beyond Cys182 and Cys277 was found to trigger cooperative modification of the remaining seven Cys residues and protein unfolding. This study highlights the power of top-down FTICR mass spectrometry for analysis of the cysteine reactivity and redox chemistry in multiple cysteine-containing proteins.

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Section: Discussionmentioning

confidence: 99%

Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

Scotcher

Clarke

Weidt

et al. 2011

J. Am. Soc. Mass Spectrom.

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“…Specific algorithms have been designed primarily for the analysis of modifications. These include FindMod [180], SALSA [181][182][183], ProSight PTM [184,185] and algorithms for the multi-dimensional LC/MS/MS [186,187]. These approaches use both mass and/or the characteristic fragmentation of the modification and amino acid to uncover modification sites.…”

Section: Discussionmentioning

confidence: 99%

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Ren

Freitas

2007

Expert Review of Proteomics

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Due to the intimate interactions between histones and DNA, the characterization of histones has become the focus of great attention. A series of mass spectrometry-based technologies have been dedicated to the characterization and quantitation of different histone forms. This review focuses on the discussion of mass spectrometry-based strategies used for the characterization of histones and their post-translational modifications. Keywordshistone; mass spectrometry; post-translational modification Histones are a group of highly conserved proteins throughout eukaryotic evolution [1]. The core histones (H4, H3, H2A and H2B) form an octamer, in which a H2A-H2B dimer associates on each side of the H3-H4 tetramer through an interaction between the C-terminal halves of H2B and H4 [2]. The negatively charged DNA superhelix wraps around the histone octamer to form repeating nucleosome cores, which further assemble into higher order chromatin fibers stabilized by the linker histones (H1 and H5) and linker DNA [3]. The histone fold regions in core histones are highly conserved α-helices, which are connected by two loops. The third histone structural motif comprises the flexible and irregular histone tails, which are extended and reach outside of the nucleosomal unit to interact with DNA [4]. Challenge of molecular characterization: post-translational modifications & sequence variationsAs a building block of the nucleosome, histones play an important role in chromatin assembly and affect the interactions between DNA and other chromatin-associated proteins. Histones regulate gene transcription through their diverse post-translational modifications (PTMs), which include lysine acetylation, lysine and arginine methylation, serine and threonine phosphorylation, lysine ubiquitination, lysine sumoylation, and poly-ADP-ribosylation of glutamic acid [5][6][7][8]. Among all the PTMs, histone acetylation and methylation have been most †

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“…The thorough high resolution analysis of spectra by Horn (THRASH) program was used to generate the experimental peak lists obtained for the ECD experiments [45]. The monoisotopic masses of fragment ions were imported into the ProSight PTM program for generation of the fragmentation summaries [46]. Mass tolerance was 0.1 Da for identifying the fragment ions.…”

Section: Discussionmentioning

confidence: 99%

Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein

Woodling

Eyler

Tsybin

et al. 2007

J. Am. Soc. Mass Spectrom.

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A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data. . This protein also binds calmodulin [7], acidic membrane phospholipids [8 -10], and actin filaments [11]. These interactions are believed to be regulated by protein phosphorylation [3,4,12,13]. The MARCKS protein is also a major substrate for protein kinase C (PKC) [14]. Protein kinase C-related kinase (PRK1) and -associated kinase also phosphorylate the phosphorylation site domain (PSD) [15,16]. PKC contains two regions: the N-terminal regulatory region and the C-terminal catalytic region. This kinase phosphorylates serine and threonine residues in basic peptide and protein sequences. Thus, PKC phosphorylates a small segment of the MARCKS protein, displacing the protein from membranes. The phosphorylation of the MARCKS protein then inhibits cross-linking activity of filamentous (F) actin, calcium-calmodulin binding, and binding to the plasma membrane [3,7,9,11,[17][18][19].The MARCKS protein contains three highly conserved regions: the phosphorylation site domain (PSD), the myristoylated amino-terminal domain, and the MARCKS homology domain. The PSD region, also known as the effector domain (ED), is the most studied, and is a basic peptide comprised of 25 amino acid residues. This region is believed to be central to the activity of the MARCKS protein.

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ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry

Cited by 183 publications

References 13 publications

Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

Identification of Two Reactive Cysteine Residues in the Tumor Suppressor Protein p53 Using Top-Down FTICR Mass Spectrometry

Mass spectrometry-based strategies for characterization of histones and their post-translational modifications

Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich c kinase protein

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