Nano-LC FTICR Tandem Mass Spectrometry for Top-Down Proteomics: Routine Baseline Unit Mass Resolution of Whole Cell Lysate Proteins up to 72 kDa

Tipton, Jeremiah D.; Tran, John; Catherman, Adam D.; Ahlf, Dorothy R.; Durbin, Kenneth R.; Lee, Ji Eun; Kellie, John F.; Kelleher, Neil L.; Hendrickson, Christopher L.; Marshall, Alan G.

doi:10.1021/ac202651v

Cited by 38 publications

(35 citation statements)

References 70 publications

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“…Besides the increase in field strength, a wired octupole following the ion trap allowed for the storage of an increased number of ions before detection in the ICR cell. This instrument was also utilized for analysis of GELFrEE fractions, using the improvement in sensitivity and resolution for the detection and identification of higher molecular weight proteins [23,100]. …”

Section: Fourier Transform Ion Cyclotron Resonance Mass Spectrometrymentioning

confidence: 99%

Top Down proteomics: Facts and perspectives

Catherman

Skinner

Kelleher

2014

Biochemical and Biophysical Research Communications

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The rise of the “Top Down” method in the field of mass spectrometry-based proteomics has ushered in a new age of promise and challenge for the characterization and identification of proteins. Injecting intact proteins into the mass spectrometer allows for better characterization of post-translational modifications and avoids several of the serious “inference” problems associated with peptide-based proteomics. However, successful implementation of a Top Down approach to endogenous or other biologically relevant samples often requires the use of one or more forms of separation prior to mass spectrometric analysis, which have only begun to mature for whole protein MS. Recent advances in instrumentation have been used in conjunction with new ion fragmentation using photons and electrons that allow for better (and often complete) protein characterization on cases simply not tractable even just a few years ago. Finally, the use of native electrospray mass spectrometry has shown great promise for the identification and characterization of whole protein complexes in the 100 kDa to 1 MDa regime, with prospects for complete compositional analysis for endogenous protein assemblies a viable goal over the coming few years.

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Section: Fourier Transform Ion Cyclotron Resonance Mass Spectrometrymentioning

confidence: 99%

Top Down proteomics: Facts and perspectives

Catherman

Skinner

Kelleher

2014

Biochemical and Biophysical Research Communications

Self Cite

420

373

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“…It also emphasizes the likelihood that this and the p53 populations examined previously comprise a substantial repertoire of proteoforms. These observations highlight the need for application of complementary methods to define the particular combinations of PTMs present on p53 molecules and to estimate their relative abundances in p53 populations exhibiting different properties, notably, analysis by "top-down" mass spectrometry (106)(107)(108).…”

Section: Discussionmentioning

confidence: 99%

Impact of the Adenoviral E4 Orf3 Protein on the Activity and Posttranslational Modification of p53

DeHart

Perlman

Flint

2015

J Virol

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Our previous studies have established that the p53 populations that accumulate in normal human cells exposed to etoposide or infected by an E1B 55-kDa protein-null mutant of human adenovirus type 5 carry a large number of posttranslational modifications at numerous residues (C. J. DeHart, J. S. Chahal, S. J. Flint, and D. H. Perlman, Mol Cell Proteomics 13:1-17, 2014, http: //dx.doi.org/10.1074/mcp.M113.030254). In the absence of this E1B protein, the p53 transcriptional program is not induced, and it has been reported that the viral E4 Orf3 protein inactivates p53 (C. Soria, F. E. Estermann, K. C. Espantman, and C. C. O'Shea, Nature 466:1076 -1081, 2010, http://dx.doi.org/10.1038/nature09307). As the latter protein disrupts nuclear Pml bodies, sites at which p53 is modified, we used mass spectrometry to catalogue the posttranscriptional modifications of the p53 population that accumulates when neither the E1B 55-kDa nor the E4 Orf3 protein is made in infected cells. Eighty-five residues carrying 163 modifications were identified. The overall patterns of posttranslational modification of this population and p53 present in cells infected by an E1B 55-kDa-null mutant were similar. The efficiencies with which the two forms of p53 bound to a consensus DNA recognition sequence could not be distinguished and were lower than that of transcriptionally active p53. The absence of the E4 Orf3 protein increased expression of several p53-responsive genes when the E1B protein was also absent from infected cells. However, expression of these genes did not attain the levels observed when p53 was activated in response to etoposide treatment and remained lower than those measured in mock-infected cells. T he cellular p53 protein was discovered by virtue of its interaction with the major product of the simian virus 40 oncogene, large T antigen (1, 2). The p53 tumor suppressor is a master regulator of cellular responses to internal and external stresses, when it can induce inhibition of cell cycle progression, apoptosis, or other responses, such as changes in metabolism. Under normal conditions, the human p53 protein is maintained at low concentrations, for example, as a result of its targeting for proteasomal degradation by the E3 ubiquitin ligase Hdm2 (3-5). Once stabilized and activated in response to genotoxic and other forms of stress, p53 binds to specific promoter sequences to activate or repress the transcription of numerous target genes (6-10) and can also operate in the cytoplasm to induce apoptosis by transcription-independent mechanisms (reviewed in references 11 to 14).One of the first interactions between human adenovirus type 5 (Ad5) and cellular proteins to be identified was the association of the viral E1B 55-kDa protein with p53 (15). In view of its crucial roles in regulating cell survival and other aspects of cellular physiology, considerable effort has since been devoted to elucidation of the impacts of adenoviral gene products on the activities and properties of p53. The viral immediate-early E1A prote...

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“…Then they are identified by MS. The results obtained in this type of studies involve the deduction of protein masses, structures, and posttranslational modifications (Tipton et al, 2012;Cannon et al, 2014).…”

Section: Protein/peptide Analysismentioning

confidence: 99%