MassIVE MSV000088805

Complete Public PXD031744

Top-down proteome of de-fatted Bos taurus milk from an animal with clinical mastitis

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Description

Quarter-milk samples were collected from a cow with clinical mastitis on day 13 and day 16 postpartum. Milk was collected from a clinical quarter with signs of abnormality (i.e., flakes, clots, blood) and one normal quarter of the same animal. Milk samples were processed with the MPLEx protocol (http://dx.doi.org/10.1039/C6AN02486F) to precipitate the proteins. The pellet was re-suspended in 500uL 3% ACN, 0.1% formic and the suspension was then either filtered with Amicon 100k Da molecular weight cutoff membrane filter, or ultracentrifuged at 100 kg for 10 min. The filtrate or the supernatant, respectively, were diluted at 0.2 ug/uL in 3% ACN, 0.1% formic acid for LCMS analysis. A Waters NanoAcquity LC was used for online separation. The binary mobile phase solvents are 0.2% formic acid in water (A), and 0.2% formic acid in ACN (B). For each LCMS experiment, 5 uL sample was injected into the trapping column (in-house packed, 5 cm, inner diameter 150 um, outer diameter 360 um, C2 reversed phase particles, MEB2-3-300, Separation Methods Technologies). After washing with 5% mobile phase B at 3 uL/min for 5 min, sample was loaded on to the C2 analytical column (in-house packed, 50 cm, inner diameter 100 um, outer diameter 360 um, same packing material as the trap column) and separated with 5-50% mobile phase B gradient at 0.3 uL/min over 100 min. Top-down MS data were collected on Orbitrap based mass spectrometers with standard data dependent acquisition. The membrane filtered samples were analyzed on a Thermo Orbitrap Eclipse with higher-energy collision dissociation for MS2. The ultracentrifuged samples were analyzed on both a Thermo Q Exactive HF (HCD for MS2) and a Thermo Orbitrap Fusion Lumos (HCD and also ETD for MS2). The main data included here are both high resolution MS1 (120k) and high resolution MS2 (60k). Lower resolution MS1 data were collected for Q Exactive HF (15k) and Lumos (7.5k) instruments. These RAWs are included but not analyzed for this deposit. The average of MS/MS counts for the Orbitrap Eclipse experiments was 22,671, greatly outnumbering those of the Orbitrap Fusion Lumos (13,408) and of the Q-Exactive HF (8,820) for the same LC duration (the numbers of spectra reflect both sample processing and instrument method differences). The Eclipse set produced an interquartile range of +6 to +13 for its precursor charges, and the Fusion Lumos reported +6 to +12. The Q-Exactive HF, however, yielded a lower interquartile range of +5 to +9. Proteomics analyses were performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the U.S. OBER and located at PNNL in Richland, Washington. PNNL is a multi-program national laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO 1830. Funding was provided by the National Institutes of Health grant number 1R01HD092297-01A1. [doi:10.25345/C52K4H] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: milk ; top-down ; phosphorylation ; mastitis

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Principal Investigators:
(in alphabetical order) 		Mark A. McGuire, University of Idaho, United States of America
Submitting User: dtabb73
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