In-depth Analysis of Tandem Mass Spectrometry Data from Disparate Instrument Types

Chalkley, Robert J.; Baker, Peter R.; Medzihradszky, Katalin F.; Lynn, Aenoch J.; Burlingame, Alma L.

doi:10.1074/mcp.m800021-mcp200

Cited by 190 publications

(171 citation statements)

References 20 publications

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“…All mass spectra were manually searched for a, b, c•/c, y, and z•/z fragment ions using Protein Prospector ver. 5.2.2 software (UCSF, San Francisco, CA, USA) [42]. Figure 1 shows the mass spectra obtained following CID and ECD of the doubly-charged peptide ions.…”

Section: Activated Ion (Ai) Ecd Photons For Infrared Irradiationmentioning

confidence: 99%

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

Jones

Mikhailov

Iniesta

et al. 2010

J. Am. Soc. Mass Spectrom.

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In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification. Here, we have investigated the electron capture dissociation (ECD) and collision-induced dissociation (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains. (J Am Soc Mass Spectrom 2010, 21, 268 -277)

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Section: Activated Ion (Ai) Ecd Photons For Infrared Irradiationmentioning

confidence: 99%

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

Jones

Mikhailov

Iniesta

et al. 2010

J. Am. Soc. Mass Spectrom.

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“…The MS method was a "top 6" data-dependent sequence with one survey scan in FT mode having mass resolution of 30,000 followed by six CID scans in LTQ targeting the first six most intense peptide ions whose m/z values were not in the dynamically updated exclusion list. The MS/MS data were searched against the SwissProt database using the in-house Protein Prospector search engine (61,62), with a concatenated database consisting of normal and randomized decoy databases (63,64). False discovery rates for phosphorylation and ubiquitination assays were estimated to be 1 and 5%, corresponding to the expectation values of 0.01 and 0.05, respectively.…”

Section: ␥-S-[mentioning

confidence: 99%

Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

Wang

Guan²,

Acharya

et al. 2011

Journal of Biological Chemistry

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Human liver CYP2E1 is a monotopic, endoplasmic reticulumanchored cytochrome P450 responsible for the biotransformation of clinically relevant drugs, low molecular weight xenobiotics, carcinogens, and endogenous ketones. CYP2E1 substrate complexation converts it into a stable slow-turnover species degraded largely via autophagic lysosomal degradation. Substrate decomplexation/withdrawal results in a fast turnover CYP2E1 species, putatively generated through its futile oxidative cycling, that incurs endoplasmic reticulum-associated ubiquitin-dependent proteasomal degradation (UPD). CYP2E1 thus exhibits biphasic turnover in the mammalian liver. We now show upon heterologous expression of human CYP2E1 in Saccharomyces cerevisiae that its autophagic lysosomal degradation and UPD pathways are evolutionarily conserved, even though its potential for futile catalytic cycling is low due to its sluggish catalytic activity in yeast. This suggested that other factors (i.e. post-translational modifications or "degrons") contribute to its UPD. Indeed, in cultured human hepatocytes, CYP2E1 is detectably ubiquitinated, and this is enhanced on its mechanismbased inactivation. Studies in Ubc7p and Ubc5p genetically deficient yeast strains versus corresponding isogenic wild types identified these ubiquitin-conjugating E2 enzymes as relevant to CYP2E1 UPD. Consistent with this, in vitro functional reconstitution analyses revealed that mammalian UBC7/gp78 and UbcH5a/CHIP E2-E3 ubiquitin ligases were capable of ubiquitinating CYP2E1, a process enhanced by protein kinase (PK) A and/or PKC inclusion. Inhibition of PKA or PKC blocked intracellular CYP2E1 ubiquitination and turnover. Here, through mass spectrometric analyses, we identify some CYP2E1 phosphorylation/ubiquitination sites in spatially associated clusters. We propose that these CYP2E1 phosphorylation clusters may serve to engage each E2-E3 ubiquitination complex in vitro and intracellularly.Hepatic cytochromes P450 (P450s) 2 are endoplasmic reticulum (ER)-anchored hemoproteins involved in the metabolism of numerous endo-and xenobiotics. These substrates can modulate P450 content, diversity, and/or function (see Refs. 1, 2 and references therein) through induction via either increased synthesis or protein stabilization, i.e. half-life prolongation (3-9). By contrast, "suicide" substrate/inactivators accelerate the degradation of certain P450s and dramatically curtail their halflives (10 -23). Such substrate-mediated P450 induction and/or enhanced turnover can influence the severity and the time course of certain pharmacokinetic/pharmacodynamic drugdrug interactions and is an important therapeutic consideration (24 -27).P450 turnover has been proposed to involve various proteolytic mechanisms (6 -9, 28 -38). However, it is now increasingly evident that in common with other type I monotopic ER proteins, P450s such as CYPs 3A (both native and structurally inactivated) undergo ER-associated degradation (ERAD) involving the ubiquitin (Ub)-dependent 26 S proteasomal system (UPS) (6 ...

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“…As detailed in the data acquisition settings above, each individual spectrum is the average of two ion transients (microscans) each. These data were then entered into Protein Prospector version 5.3.2 (26), and a search was performed against the Mus musculus entries in the UniProtKB database (downloaded on July 7, 2009 with 60,970 entries). Precursor and fragment mass tolerances of 30 ppm were allowed.…”

Section: Methodsmentioning

confidence: 99%

High Resolution Electron Transfer Dissociation Studies of Unfractionated Intact Histones from Murine Embryonic Stem Cells Using On-line Capillary LC Separation

Eliuk

Maltby

Panning

et al. 2010

Molecular & Cellular Proteomics

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Epigenetic regulation of chromatin is dependent on both the histone protein isoforms and state of their post-translational modifications. The assignment of all post-translational modification sites for each individual intact protein isoform remains an experimental challenge. We present an on-line reversed phase LC tandem mass spectrometry approach for the separation of intact, unfractionated histones and a high resolution mass analyzer, the Orbitrap, with electron transfer dissociation capabilities to detect and record accurate mass values for the molecular and fragment ions observed. From a single LC-electron transfer dissociation run, this strategy permits the identification of the most abundant intact proteins, determination of the isoforms present, and the localization of post-translational modifications. Molecular & Cellular Proteomics 9:824 -837, 2010.Over the last two decades, the integration of the unanticipated discovery of new modes of internal energy deposition and rapid technological progress in mass spectrometrybased instrument platforms has revolutionized our experimental capabilities to effectively study the inherent complexity of human and other mammalian proteomes. This includes the rapid identification of proteins, the detection and unambiguous assignment of protein isoforms, and the detection and localization of post-translational modifications (PTMs). 1The vast majority of mass spectrometry-based studies in protein biology and proteomics has used proteolytic digestion using trypsin and analysis of the resulting peptide mixtures by a variety of MS and MS/MS approaches (1-4). Although of considerable use for many purposes, this common strategy has a variety of limitations especially from the view of acquiring the information needed to establish and understand biological function. These limitations include the loss of information regarding the nature of protein isoforms and a loss of knowledge of the global presence of multiple PTMs that can co-occur at differing sites on the same protein molecule. These deficiencies are exacerbated when the portions of protein sequence coverage observed in a digest are low because peptides that would reveal the modifications or isoform-specific sequence variances may not have been detected. Additionally, the sequence identification for fragment ion spectra representing peptides with modifications that are unanticipated can be difficult to assign using common search algorithms.Hence, structural information covering an entire protein sequence has been lacking, and although needed from a protein biology viewpoint all along, the kind of methodology that would be required to obtain this knowledge has begun to emerge only recently. This has come about through the discovery of electron capture by polyprotonated peptide and protein species (5) and the more recent development of electron transfer by their reaction with suitable radical anions (6). Because these physicochemical processes deposit sufficient internal energy at the sites of charge reduction to cleave peptid...

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In-depth Analysis of Tandem Mass Spectrometry Data from Disparate Instrument Types

Cited by 190 publications

References 20 publications

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

Electron capture dissociation mass spectrometry of tyrosine nitrated peptides

Ubiquitin-dependent Proteasomal Degradation of Human Liver Cytochrome P450 2E1

High Resolution Electron Transfer Dissociation Studies of Unfractionated Intact Histones from Murine Embryonic Stem Cells Using On-line Capillary LC Separation

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