br MALDI TOF profiling of whole sperm cells

MALDI-TOF profiling of whole sperm p2y receptor and sub-cellular fractions All samples were analyzed by a MALDI-TOF mass spectrometer (Waters Corporation, Micromass Ltd., Manchester, UK) operating in positive linear mode as previously described [1]. Spectral profiles were collected in the 2000–20,000m/z mass range. Data processing was performed using MassLynx™ 4.0 software. To increase mass accuracy, internal calibration was performed. Thus, the major unknown 6797m/z constant was used as “lock mass” for all spectra. Intact spermatozoa and corresponding detergent-soluble and -insoluble extracts obtained from 4 epididymal regions (E2, E4, E6 and E9) of both epididymes from 4 animals were analyzed by MALDI-TOF MS, with 8 replicates for each of the three sample preparations. A total of 768 spectra were generated in this study. The spectra were analyzed by Progenesis MALDI™ 1.2 software (NonLinear Dynamics) as previously described [1]. After alignment of all spectra, a total of 253m/z peaks were detected (Supplementary Table 1). A total of 135m/z molecular species were characterized by Intact Cell MALDI-MS and 118m/z were newly observed by MALDI MS from SD and ID fractions.
Quantitative analysis linked to the sperm maturation process In order to characterize peak differences between epididymal spermatozoa, the intensity of each normalized peak was subjected to one way analysis of variance with Progenesis (factor epididymal regions) and to three-way analysis of variance with R software (factors being animals, epididymis, epididymal regions, the replicate interactions being the residuals), as previously described [1]. All peak signals with a differential fold-change greater than two average values of normalized intensity and a p value<0.05 were selected (Supplementary Table 1). Thus 89m/z peaks were retained for intact cells (IC), 112m/z peaks for detergent-soluble extracts (SD) and 59m/z peaks for detergent-insoluble extracts (ID) for a total of 172 unique m/z peaks (Supplementary Table 1).
Top-down mass spectrometry Identification of peptidoforms and proteoforms (endogenous species) was achieved by acquiring nano-ESI tandem high resolution mass spectrometry (MS and MS/MS). Molecular species were previously extracted with 1% and 5% formic acid from IC (region 2 or 9) or from ID fraction (region 9), desalted and concentrated using ZipTip C4 (Millipore Corporation, Billerica, MA). Eluted peptides and proteins were directly analyzed using a LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Germany) operating in positive mode, as previously described (1). Data were acquired using Xcalibur software v2.1 (Thermo Fisher Scientific, San Jose, CA). All analyses were performed manually using a high-high strategy, meaning that a MS spectrum in the 400–2000m/z mass range was followed by a MS/MS spectrum obtained by High energy Collisional Dissociation (HCD). Thus 412m/z corresponding to 217 non-redundant molecular ions were selected to induce HCD fragmentation. Identification and structural characterization were performed using ProSight PC software 2.0 (Thermo Scientific, San Jose). Raw data files were processed by THRASH (signal/noise: 2–3), and data were compared to a simple annotated “Sus scrofa” house database generated from NCBInr using Proteome Discoverer (Thermo Fisher Scientific). Automated searches were performed using the “Absolute Mass and Biomarker” search options. The mass tolerances were set at 5ppm for the monoisotopic precursors, 5Da for the average precursor and 15ppm for fragment ions mass tolerance. Disulfide modifications and N-terminal post-translation modifications (acetylation and initial methionine cleavage) were activated. Post-translational modifications such as phosphorylation and disulfide bridges were confirmed using the manual Single Protein mode. Proposed sequences with E-value<1×10−2 were considered positively identified with a minimum of 10 matching fragment ions. The data were deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [3,4] with the dataset identifier PXD001303.