Western blot quality control checks

2.3.4.1 Increased abundance of heterodimeric clusterin in gorilla seminal plasma prevents it from being an effective extracellular loading control across hominid seminal plasma

Currently there is no universal loading control for seminal plasma, like β- actin, or other housekeeping proteins, in intracellular experiments. We searched for a protein that was similar in expression across primate seminal plasma, which could act as a loading control between species. After reviewing previous shotgun Liquid Chromatography Tandem Mass Spectrometry (LC/MS- MS) data comparing human and chimpanzee seminal plasma (Chovanec et al., unpublished), two candidate proteins, albumin and clusterin, were both highly abundant and similar in

concentration between species. Both albumin and clusterin have seven amino acid differences between human and chimpanzee (Appendix A.2, Alignments 1 and 2). Clusterin was chosen because it was less likely to have false signal compared to albumin. Bovine serum albumin is used as a blank in LC/MS-MS and is in many blocking buffers, which could provide additional signal for human albumin in an experiment. When human and bovine serum albumin were aligned there were four fragments greater than six amino acids that could be cross identified between human and bovine serum albumin (Appendix A.2, Alignment 3).

Initially, human and chimpanzee seminal plasma (10µg and 20µg) was Western blotted for clusterin. Human clusterin detection was 30% higher than chimpanzee (opposite of the LC/MS-MS data). Both human and chimpanzee detected protein was the appropriate molecular weight for clusterin (Figure 2.6:A) in its monomeric form (~48kDa). When human, chimpanzee, and gorilla seminal plasma were Western blotted the results were less clear (Figure 2.6:B). Gorilla seminal plasma inconsistently blots both the monomeric form and the heterodimeric disulfide cross-linked form of clusterin (70-80kDa), which provides a challenge to quantify

clusterin signal. Human clusterin detection was 30% higher than chimpanzee and gorilla was the lowest signal, which is opposite of my LC/MS-MS discussed later in this chapter.

Figure 2.6: Clusterin is not a reliable extracellular loading control

Anti-clusterin antibody from ABCAM was used to detect clusterin in human, chimpanzee, and gorilla seminal plasma. A) Lanes 2 and 3 have 10µg of human or chimpanzee seminal plasma loaded and lanes 4 and 5 have 20µg of the same human or chimpanzee seminal plasma loaded. Human signal was roughly 30% higher than chimpanzee in both loading conditions. B) Two individuals of each species’ seminal plasma (20 µg) was loaded and detected with anti-clusterin antibody. Monomeric clusterin protein should be around 48kDa; however, heterodimeric clusterin which is disulfide cross-linked is expected to be around 70-80kDa. Though gorilla clusterin detection looks different than human and chimpanzee, it most likely has both monomeric and heterodimeric forms of clusterin.

2.3.4.2 Majority of proteolytic degradation of proteins, particularly the semenogelins, occurs before we receive semen samples

The semenogelins are known substrates for cross-linking and degradation, which explains the appearance of multiple bands in my Western blots detecting either protein (i.e., Figure 2.3). Protease inhibitor cocktails were added to semen samples (BCl-, apo, benzamide, and DMSF – “CPI-Hu#5” or Sigma mammalian protease inhibitors- “SPI-Hu#5”) to determine if a majority of protein degradation could be prevented. Semen with and without inhibitors was centrifuged to separate seminal plasma proteins from spermatozoa, and then aliquoted into different tubes and each tube was incubated at 4°C, 23°C, and 37°C for an hour, potentially allowing enzymatic

kDa 220 120 100 80 60 50 40 30 20 220 120 100 80 60 50 40 30 20

Hu#2 Ch#2 Hu#2 Ch#2 Hu#2 Hu#3 Ch#2 Ch#3 Go#2 Go#3

Anti-Clusterin

activity. Samples were quantified (Figure 2.7), and samples with protease inhibitors had a slightly higher concentration than without inhibitors. However, there were minimal differences in protein concentration across the different aliquots incubated at different temperatures.

Figure 2.7: No significant differences in protein concentration in seminal plasma with and without protease inhibitors

Human semen (Hu#5), human semen with a mix of protease inhibitors (CPI), and human semen with a commercial sigma protease inhibitor (SPI) cocktail were aliquoted and incubated at different temperatures, and their

absorbencies were measured using BioRad protein assay dye. Standard BSA samples in the Bradford assay were plotted (diamonds) in R and a linear regression line was calculated (y= 0.661x + 0.239, R2_{=0.94). The average}

calculated concentration of each sample is listed on the right and their concentrations were not significantly different (p=0.124; see Appendix A.1.4).

There were no noticeable visual differences between samples (inhibitor vs. non-inhibitor or across temperature incubation) when samples were separated by SDS-PAGE (Figure 2.8) or blotted with anti-SEMG antibodies (Figure 2.9). Both anti-SEMG1 and SEMG2 antibodies detected proteins at their respective full-length molecular weights; however, they also had smaller portions of protein detected. Essentially, a large amount of degradation occurs before we receive our semen samples. Although adding protease inhibitors may prevent subsequent

degradation, unless inhibitors are immediately added during collection, majority of proteins susceptible to proteolytic cleavage will be degraded.

Sample Concentration

Human #5 8.56 mg/mL

CPI –Hu#5 10.82 mg/mL

Figure 2.8: Seminal plasma samples with and without protease inhibitors look similar Human semen was aliquoted and immediately inhibitors (BCL-, apo, benzamide, and DMSF) were added (inhibitor cocktail/CPI) or a premade mammalian protease inhibitor cocktail (Sigma inhibitor mix/SPI) was added.

Centrifugation removed sperm cells and seminal plasma was subjected to varying temperature (4°C, 23°C, or 37°C) incubations for an hour. Seminal plasma with and without protease inhibitors visually look the same when stained with coomassie.

Figure 2.9: SEMG1 and SEMG2 are degraded before addition of protease inhibitors Human semen was aliquoted and immediately inhibitors (BCL-, apo, benzamide, and DMSF) were added (inhibitor cocktail/CPI) or a premade mammalian protease inhibitor cocktail (Sigma inhibitor mix/SPI) was added.

Centrifugation removed sperm cells and seminal plasma was subjected to varying temperature (4°C, 23°C, or 37°C) incubations for an hour. There were minimal or no differences of anti-SEMG binding between 10µg seminal plasma with and without protease inhibitors. Both full length and varying sizes of degraded SEMG peptides were detected across samples.

Hu#5 CPI – Hu#5 SPI –Hu#5 No inhibitors Inhibitor cocktail Sigma Inhibitors

4°C 23°C 37°C 4°C 23°C 37°C 4°C 23°C 37°C kDa 198 98 62 49 38 27 17 Anti-SEMG1 Anti-SEMG2

Hu#5 CPI- Hu#5 SPI- Hu#5

No inhibitors Inhibitor cocktail Sigma Inhibitors

4°C 23°C 37°C 4°C 23°C 37°C 4°C 23°C 37°C

Hu#5 CPI- Hu#5 SPI- Hu#5

No inhibitors Inhibitor cocktail Sigma Inhibitors 4°C 23°C 37°C 4°C 23°C 37°C 4°C 23°C 37°C kDa 220 120 80 60 50 40 30 20 kDa 220 120 80 60 50 40 30 20

2.3.5 LC/MS-MS and Western blot identification of seminal plasma proteins among