SLeX-BSA and LeX-BSA IP sperm protein-summary (MALDI-TOF-MS/MS)

Identification of Sialyl-Lewis(x) (SLeX)/ Lewis(x)
(LeX)-binding proteins from human sperm membrane protein extracts using
SLeX/LeX-BSA affinity chromatography followed by MALDI-TOF-MS/MS. SLeX-binding
proteins on the plasma membrane of capacitated spermatozoa were identified by
our established chromatographic method using SLeX-BSA neoglycoprotein affinity
column followed by mass spectrometric analysis as described. In brief,
capacitated spermatozoa (100×106) were washed thrice in EBSS.
Non-integral, peripheral membrane-associated proteins on spermatozoa were
removed by incubation of the washed spermatozoa in 1 M NaCl in PBS with gentle
stirring for 10 minutes at 25ºC. The spermatozoa were then collected by
centrifugation at 600× g for 10 minutes before extraction of the sperm plasma
membrane proteins by the ProteoExtract Native Membrane Protein Extraction Kit
(Merck, Kenilworth, NJ) according to the manufacturer’s instructions. The
insoluble fraction was discarded after centrifugation at 15,000× g for 40
minutes. The supernatant was diluted in a solution of MOPS-NaOH buffer (pH 7.3)
containing 0.2% Triton X-100 and 6 mM MnCl2. SLeX-BSA (Dextra, Reading, UK)
conjugated sepharose beads (GE Healthcare) were used to precipitate the
SLeX-binding protein from the extracted membrane protein fractions. LeX-BSA,
which did not bind to human spermatozoa was used as a control.

采用SLeX/LeX-BSA亲和层析法，结合基质辅助激光解吸电离飞行时间质谱/串联质谱（MALDI-TOF-MS/MS）技术，从人类精子膜蛋白提取物中鉴定Sialyl-Lewis(x) (SLeX)/ Lewis(x)结合蛋白。通过建立的色谱方法，利用SLeX-BSA偶联亲和层析柱，对精子细胞质膜上的SLeX结合蛋白进行鉴定，并辅以质谱分析，具体操作方法如下所述。简而言之，将精子细胞在EBSS缓冲液中清洗三次（100×10^6）。通过在25℃下用1 M NaCl在磷酸盐缓冲盐溶液（PBS）中温和搅拌10分钟，去除精子细胞上的非整蛋白、外周膜结合蛋白。随后，以600× g的转速离心10分钟收集精子细胞，然后按照制造商的说明，使用ProteoExtract原生膜蛋白提取试剂盒（Merck，Kenilworth，NJ）提取精子质膜蛋白。经15,000× g离心40分钟后，弃去不溶性部分。上清液用含有0.2% Triton X-100和6 mM MnCl2的MOPS-NaOH缓冲液（pH 7.3）稀释。利用SLeX-BSA偶联的琼脂糖珠（GE Healthcare）从提取的膜蛋白组分中沉淀SLeX结合蛋白。未结合人类精子细胞的LeX-BSA作为对照。