Differential extraction and enrichment of human sperm surface proteins in a proteome: Identification of immunocontraceptive candidates

Abstract

The objective of this study was to discover previously unknown human sperm surface proteins that may be candidate contraceptive vaccinogens. To this end, methods of concentrating human sperm proteins for microsequencing by mass spectrometry were used, which increased the likelihood of identifying surface proteins. Vectorial labeling, differential extraction and two-dimensional (2-D) gel electrophoresis were employed to identify and isolate proteins accessible at the cell surface. Percoll harvested or swim-up sperm were either solubilized directly or solubilized after surface labeling with sulfo-succinimidyl-6-(biotinamido)hexanoate (sulfo-NHS-LC-biotin). Comparisons were made of proteins extracted with four lysis buffers: (i) Celis buffer containing 9.8 M urea and 2% Igepal CA-630; (ii) 1% Triton X (TX)-100; (iii) 1.7% TX-114 followed by phase partitioning; or (iv) 1 M NaCl. Blots of proteins separated by high-resolution 2-D electrophoresis were probed with avidin and antibodies to known proteins specific for three domains: the sperm surface (SAGA-1), the acrosome (SP-10), and the cytoskeleton (alpha-tubulin). Celis buffer (45 min) extracted proteins from all three major compartments. However, a 20-s extraction in Celis buffer enriched for several proteins and enabled the identification of several novel peptides by mass spectrometry. Mild extraction with TX-100 or 1 M NaCl solubilized mainly membrane and acrosomal proteins, but not cytoskeletal proteins. Comparison of biotinylated proteins extracted by each method showed that the major vectorially labeled proteins solubilized by Celis buffer were also solubilized by TX-100, TX-114, and 1 M NaCl. Extraction with TX-114 followed by phase-partitioning significantly enriched hydrophobic surface proteins and aided resolution and isolation. Eight protein spots microsequenced following all these extraction methods proved to be novel sperm molecules.

SAMP14, a Novel, Acrosomal Membrane-associated, Glycosylphosphatidylinositol-anchored Member of the Ly-6/Urokinase-type Plasminogen Activator Receptor Superfamily with a Role in Sperm-Egg Interaction

“…Triton X-114 phase partitioning was employed to enrich potential membrane proteins from human spermatozoa before or after surface labeling of sperm with sulfo-NHS-LCbiotin. Biotinylation of sperm was performed by vectorial labeling conditions described previously (10). To enrich for hydrophobic membrane proteins, Triton X-114 phase separation was performed as described by Bordier (12) and modified by Shetty et al (10).…”

“…Biotinylation of sperm was performed by vectorial labeling conditions described previously (10). To enrich for hydrophobic membrane proteins, Triton X-114 phase separation was performed as described by Bordier (12) and modified by Shetty et al (10). Two-dimensional gel electrophoresis was performed according to the method of O'Farrell et al (13), with modifications (10).…”

“…Gels were stained with Coomassie Blue or blotted. For Western blots, proteins were transferred to nitrocellulose membrane and processed as described previously (10).…”

“…However, membrane molecules represent only a small subset of proteins in total cellular extracts, and specialized methods are required for their enrichment, isolation, and characterization. To increase the likelihood of identifying sperm membrane molecules that may be involved in the acrosome reaction and/or sperm-egg interaction, we adopted the strategy of Triton X-114 phase partitioning prior to two-dimensional gel electrophoresis and microsequencing by mass spectrometry (10). Several spots in this previous study yielded novel peptide sequences.…”

Characterization of 3-hydroxyisobutyrate dehydrogenase, HIBADH, as a sperm-motility marker

“…This approach has been used specifically to discover differential sperm-related proteins (e.g., sperm-surface proteins and antisperm antibodies) [21][22][23]. In other studies, calcium-binding proteins and proteins undergoing tyrosine phosphorylation during capacitation were identified using the proteomic approach [24,25].…”