Estimating relative abundances of proteins from shotgun proteomics data

McIlwain, Sean J.; Mathews, Michael; Bereman, Michael S.; Rubel, Edwin W.; MacCoss, Michael J.; Noble, William Stafford

doi:10.1186/1471-2105-13-308

Cited by 92 publications

(69 citation statements)

References 19 publications

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“…Proteomic analysis of the specific biotin-tagged S. cerevisiae proteins identified a number of proteins that were bound by streptavidin agarose. Since it is impractical to quantitatively rank protein hits based on mass spectral data alone in ‘shotgun’ proteomic experiments [59], the identified proteins were sorted by topological analysis (TMHMM server available through the Center for Biological Sequence Analysis at http://www.cbs.dtu.dk/services/TMHMM-2.0/) and annotation of subcellular location available through the UniProt Consortium (www.uniprot.org). The results of these experiments are summarized in Table 3.…”

Section: Resultsmentioning

confidence: 99%

Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells

Rush

Subramanian

et al. 2016

CCB

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Background Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified. Methods The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen “click” chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates. In vitro enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway. ER proteins that photoreacted with the novel probes were identified by MS. Results The potential of the newly designed photoprobes, m-PAL-Cit-P and p-PAL-Cit-P, for identifying previously unidentified Dol-P-interacting proteins is supported by the observation that they are enzymatically mannosylated by Man-P-Dol synthase (MPDS) from Chinese Hamster Ovary (CHO) cells at an enzymatic rate similar to that for Dol-P. MS analyses reveal that DPM1, ALG14 and several other yeast ER proteins involved in DLO biosynthesis and lipid-mediated protein O-mannosylation photoreacted with the novel probes. Conclusion The newly-designed photoprobes described in this paper provide promising new tools for the identification of yet to be identified Dol-P interacting ER proteins in yeast and mammalian cells, including the Dol-P flippase required for the “re-cycling” of the glycosyl carrier lipid from the lumenal monolayer of the ER to the cytoplasmic leaflet for new rounds of DLO synthesis.

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Section: Resultsmentioning

confidence: 99%

Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells

Rush

Subramanian

et al. 2016

CCB

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show abstract

“…By comparison, all methods showed good reproducibility ( R 2 >0.95) for higher abundance proteins (Supplemental Figure 1), which is in line with our previous reports. 23,26,42,43 As a result, in this study the ICB was employed to investigate the molecular mechanism of OIR.…”

Section: Resultsmentioning

confidence: 99%

Proteomic Profiling of the Retinas in a Neonatal Rat Model of Oxygen-Induced Retinopathy with a Reproducible Ion-Current-Based MS1 Approach

Beharry

Shen

et al. 2015

J. Proteome Res.

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Investigation of the retina proteome during hypoxia-induced retinal neovascularization is valuable for understanding pathogenesis of retinopathy of prematurity (ROP). Here we employed a reproducible ion-current-based MS1 quantification approach (ICB) to explore the retinal proteomic changes in early stage of ROP in a rat model of oxygen-induced retinopathy (OIR). Retina proteins, which are rich in membrane proteins, were efficiently extracted by a detergent-cocktail and subjected to precipitation/on-pellet-digestion, followed by nano-LC-MS analysis on a 75-cm column with a 7-h gradient. The high reproducibility of sample preparation and chromatography separation enabled excellent peak alignment and contributed to the superior performance of ICB over parallel label-free approaches. In this study, sum-of-intensity with rejection was incorporated to determine the protein ratios. In total, 1325 unique protein groups were quantified from rat retinas (n = 4/group) with at least two distinct peptides at a protein FDR of 1%. Thirty-two significantly altered proteins were observed with confidence, and the elevated glial fibrillary acidic protein and decreased crystalline proteins in OIR retinas agree well with previous studies. Selected key alterations were further validated by Western blot analysis. Interestingly, Rab21/RhoA/ROCK2/moesin signaling pathway was found to be involved in retinal neovascularization of OIR. Moreover, highly elevated annexin A3, a potential angiogenic mediator, was observed in OIR retinas and may serve as a potential therapeutic target. In conclusion, reproducible ICB profiling enabled reliable discovery of many altered mediators and pathways in OIR retinas, thereby providing new insights into molecular mechanisms involved in pathogenesis of ROP.

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“…N-AgrD peptides were produced by three USA300 lineage MRSA, two ST59 MRSA (Huang et al, 2012), and clonal colony ST45 of USA600 MRSA lineage ( Figure 1C ). In successive MS experiments, peptide sequences most abundantly mapped to AgrD (McIlwain et al, 2012), herein designated (i) N-AgrD F20, (ii) N-AgrD F24, and (iii) N-AgrD D20, were synthesized and isolated at >95% purity for functional assessment.…”

Section: Resultsmentioning

confidence: 99%

N-Terminal ArgD Peptides from the Classical Staphylococcus aureus Agr System Have Cytotoxic and Proinflammatory Activities

Gonzalez¹,

Corriden²,

Akong-Moore³

et al. 2014

Chemistry & Biology

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SUMMARY AgrD is the precursor for the auto-inducing peptide in a quorum sensing system regulating virulence phenotypes of the pre-eminent pathogen Staphylococcus aureus. Mass spectrometry-based methods, including molecular networking, identified formylated and non-formylated peptide variants derived from the AgrD N-terminal leader domain in S. aureus cell-free culture supernatants. Functional assessment of these peptides revealed the unexpected bioactivities including human cell-line cytotoxicity, modulation of neutrophil chemotaxis, neutrophil extracellular trap formation, and the aggravation of skin lesions in vivo.

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Estimating relative abundances of proteins from shotgun proteomics data

Cited by 92 publications

References 19 publications

Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells

Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells

Proteomic Profiling of the Retinas in a Neonatal Rat Model of Oxygen-Induced Retinopathy with a Reproducible Ion-Current-Based MS1 Approach

N-Terminal ArgD Peptides from the Classical Staphylococcus aureus Agr System Have Cytotoxic and Proinflammatory Activities

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