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- Origin of Ram Epididymal Fluid Exosome-Like Vesicles: RESULTS(3)

 

All the characteristic proteins of these latter pellets were absent from the pellet of the vasectomized animals. In contrast, the pattern obtained from the epididymal vesicles and the seminal plasma vesicles from the normal animals differed only by two protein bands at 14 and 55 kDa.

Identification of the Vesicle Proteins by Two-Dimensional SDS-PAGE, Mass Spectrometry, and Western Blotting

The vesicle proteins from the caudal fluid (Fig. 4) and from the seminal plasma (not shown) were separated by two-dimensional gel electrophoresis to allow mass spectrometry identification. Both preparations gave identical protein profiles except for two series of spots at about 14 and 55 kDa present only in the seminal plasma (see also Fig. 3B). The major protein spots from the vesicles observed by Coomassie-blue staining (Fig. 4) were processed for identification by mass spectrometry. Two different approaches were used: Maldi-TOF, which allowed comparison of the tryptic pattern of a protein with the theoretical tryptic pattern in databases; and nano-HPLC-MS-MS, which allowed sequencing of specific tryptic fragments and search for homology in data bases (see Tables 1 and 2).

TABLE 1. Names, accession numbers, size, sequence of the peptides obtained by LC-MS-MS, and number of peptides and percentage of the protein coverage obtained by Maldi for each protein described in Figure 3.
table1Identification, Proteomic Profiling-4

TABLE 2.
table2Identification, Proteomic Profiling-5

TABLE 3.
table3Identification, Proteomic Profiling-6

Fig4Identification, Proteomic Profiling-7
FIG. 4. Two-dimensional gel electrophoresis of the vesicle proteins. The vesicles were treated and separated by two-dimensional gel electrophoresis under denaturing conditions. The spots were cut and processed by mass spectrometry to identify the proteins (see also Table 1). 6-16% SDS-PAGE; silver-stained gel.

March 1, 2014 Epididymal
Tags: epididymis epididymosomes exosomes gamete biology