A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC)

Orton, Dennis J.; Doucette, Alan A.

doi:10.1016/j.jchromb.2013.01.021

Cited by 8 publications

(7 citation statements)

References 20 publications

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“…Mass spectrometry was on an LTQ classic linear ion trap (ThermoFisher, San Jose, CA, USA), coupled to an Agilent 1200 HPLC system. Digested protein fractions were desalted by offline reversed phase HPLC with UV detection [ 26 ], loading one-third of the total volume of purified protein onto a 75 µm × 30 cm self-packed C12 column (3 µm Jupiter beads, Phenomenex, Torrance, CA, USA). Peptides were separated using a 1 h linear solvent gradient from 5% acetonitrile/water/0.1% formic acid to 35% acetonitrile/water/0.1% formic acid at flow rate of 0.25 µL/min.…”

Section: Methodsmentioning

confidence: 99%

Differential Proteome Analysis of Extracellular Vesicles from Breast Cancer Cell Lines by Chaperone Affinity Enrichment

et al. 2017

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The complexity of human tissue fluid precludes timely identification of cancer biomarkers by immunoassay or mass spectrometry. An increasingly attractive strategy is to primarily enrich extracellular vesicles (EVs) released from cancer cells in an accelerated manner compared to normal cells. The Vn96 peptide was herein employed to recover a subset of EVs released into the media from cellular models of breast cancer. Vn96 has affinity for heat shock proteins (HSPs) decorating the surface of EVs. Reflecting their cells of origin, cancer EVs displayed discrete differences from those of normal phenotype. GELFrEE LC/MS identified an extensive proteome from all three sources of EVs, the vast majority having been previously reported in the ExoCarta database. Pathway analysis of the Vn96-affinity proteome unequivocally distinguished EVs from tumorigenic cell lines (SKBR3 and MCF-7) relative to a non-tumorigenic source (MCF-10a), particularly with regard to altered metabolic enzymes, signaling, and chaperone proteins. The protein data sets provide valuable information from material shed by cultured cells. It is probable that a vast amount of biomarker identities may be collected from established and primary cell cultures using the approaches described here.

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Section: Methodsmentioning

confidence: 99%

Differential Proteome Analysis of Extracellular Vesicles from Breast Cancer Cell Lines by Chaperone Affinity Enrichment

et al. 2017

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“…Following incubation, 10 μL (equivalent to 10 μg E. coli protein) was subject to RP HPLC to remove the formic acid. A self‐packed 10 cm × 1 mm R2 column was employed (20 μm beads, Applied Biosystems, Burlington, Canada), using a combined temperature/solvent gradient described previously . In brief, the mobile phase was increased over an 8 min period from 5 to 80% acetonitrile/water with 0.1% TFA, while the column temperature was ramped from 25 to 80 o C in order to collect the intact protein as a single fraction.…”

Section: Methodsmentioning

confidence: 99%

Preventing N‐ and O‐formylation of proteins when incubated in concentrated formic acid

Zheng

Doucette

2016

Proteomics

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Concentrated formic acid is among the most effective solvents for protein solubilization. Unfortunately, this acid also presents a risk of inducing chemical modifications thereby limiting its use in proteomics. Previous reports have supported the esterification of serine and threonine residues (O-formylation) for peptides incubated in formic acid. However as shown here, exposure of histone H4 to 80% formic (1 h, 20(o) C) induces N-formylation of two independent lysine residues. Furthermore, incubating a mixture of Escherichia coli proteins in formic acid demonstrates a clear preference toward lysine modification over reactions at serine/threonine. N-formylation accounts for 84% of the 225 uniquely identified formylation sites. To prevent formylation, we provide a detailed investigation of reaction conditions (temperature, time, acid concentration) that define the parameters permitting the use of concentrated formic acid in a proteomics workflow for MS characterization. Proteins can be maintained in 80% formic acid for extended periods (24 h) without inducing modification, so long as the temperature is maintained at or below -20(o) C.

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“…The membrane-enriched E. coli protein fractions were analyzed on the LTQ instrument but as intact proteins (i.e., omitting tryptic digestion). Formic acid was removed from the sample by loading the recovered extract onto a self-packed 1 × 50 mm R2 column (Applied Biosystems) using a temperatureprogrammed gradient described previously by Orton et al, 48 recovering the intact protein as a single fraction. Following partial solvent evaporation, the equivalent of 1 μg total protein was then loaded onto self-packed 100 μm × 100 mm Magic C4 column (300 Å, 5 μm, Michrom Bioresources, Auburn, CA), interfaced to a 75 μm Nanospray Tip (New Objective, Woburn, MA).…”

Section: Sds Removalmentioning

confidence: 99%

Automated SDS Depletion for Mass Spectrometry of Intact Membrane Proteins though Transmembrane Electrophoresis

et al. 2016

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Membrane proteins are underrepresented in proteome analysis platforms because of their hydrophobic character, contributing to decreased solubility. Sodium dodecyl sulfate is a favored denaturant in proteomic workflows, facilitating cell lysis and protein dissolution; however, SDS impedes MS detection and therefore must be removed prior to analysis. Although strategies exist for SDS removal, they provide low recovery, purity, or reproducibility. Here we present a simple automated device, termed transmembrane electrophoresis (TME), incorporating the principles of membrane filtration, but with an applied electric current to ensure near-complete (99.9%) removal of the surfactant, including protein-bound SDS. Intact proteins are recovered in solution phase in high yield (90-100%) within 1 h of operation. The strategy is applied to protein standards and proteome mixtures, including an enriched membrane fraction from E. coli, resulting in quality MS spectra free of SDS adducts. The TME platform is applicable to both bottom-up MS/MS as well as LC-ESI-MS analysis of intact proteins. SDS-depleted fractions reveal a similar number of protein identifications (285) compared wit a non-SDS control (280), being highly correlated in terms of protein spectral counts. This fully automated approach to SDS removal presents a viable tool for proteome sample processing ahead of MS analysis. Data are available via ProteomeXchange, identifier PXD003941.

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A universal, high recovery assay for protein quantitation through temperature programmed liquid chromatography (TPLC)

Cited by 8 publications

References 20 publications

Differential Proteome Analysis of Extracellular Vesicles from Breast Cancer Cell Lines by Chaperone Affinity Enrichment

Differential Proteome Analysis of Extracellular Vesicles from Breast Cancer Cell Lines by Chaperone Affinity Enrichment

Preventing N‐ and O‐formylation of proteins when incubated in concentrated formic acid

Automated SDS Depletion for Mass Spectrometry of Intact Membrane Proteins though Transmembrane Electrophoresis

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