Analysis Of Proteomics In Seminal Plasma Of Male Infertility Human By Surface Enhanced Laser Desorption/Ionization-Time Of Flight Mass Spectrometry

Male factor infertility is the most common defined cause in infertile couples.According to the report of World Health Organization (WHO), Among 80 millioncouples suffered with infertility, in which, male factor is solely responsible in about40%, whereas women' factors and double couples' factors were 50% and 10%respectively. In China, a survey in Hebei province showed that the infertile malepersons reached 33.15% among the 39,476 child-bearing age couples. The reasons formale infertility are variable and complexes, which might be associated with genetic orchromosomal abnormalities, dysfunction or abnormal structure of reproductivesystem, alteration of hormone regulation, infection of reproductive duct, immunologyfactor and so on. However, gene expression is only one aspect of the complexregulatory network. It has been estimated that every transcribed gene produces morethan one translated proteins, and even further variation derives from posttranslationalmodifications and protein-protein interactions. Furthermore, analysis of geneexpression does not readily predict protein abundance or supply information about protein function at the biochemical level, with each protein participating in numerousdistinct biochemical pathways.The classic proteomics approach involves two-dimensional polyacrylamide gelelectrophoresis. By which, proteins are separated in two sequential steps, isoelectricfocusing (IEF) in accordance with their charges and sodium dodecyl sulphate (SDS)in accordance with their molecular weights. But the technique lacks robustness,proteins with low molecular masses or low abundance can be underrepresented anddifficult to distinguish.SELDI-TOF-MS (surface-enhanced laser desorption/ionization time of flightmass spectrometry) is one of the recently developed technologies, based on capturingproteins/peptides by chemically modified surface, it is specifically powerful foranalyzing the complex biological samples. SELDI offers the advantages of simple,rapid, high throughput examination as well as high specificity and sensitivity, enablesthe comparative analysis of virtually any given protein-containing solution by using only minuteamount of clinical samples without destroying the proteins, which are not available forconventional methods. Furthermore, reduction in sample volume improved the abilityto detect lower abundance proteins. Comparing with two-dimensional polyacrylamidegel electrophoresis, the outstanding advantage of SELDI is for profiling low-massproteins/peptides.Despite physiological and medical interest in seminal plasma, previous studiestrying to cover large numbers of proteins fell short of providing in-depth andhigh-confidence identifications of seminal proteins. Methods based on 2D gelelectrophoresis revealed many protein spots, as well as quantitative changes in normaland impaired spermatogenesis, but only a very small number of identified proteins.So comparative proteomics study to determine whether human seminal plasmacontained proteins associated with male infertility is a flash thought. More over, exhibit the relationship of protein-protein interaction and mechanism of key protein inmale infertility has significantly value for later research.In this study, we have developed a system using SELDI-TOF-MS that is capableof analyzing the proteome of seminal plasma of many kinds of infertility male. Thegoal of this work was to identify differentially expressed proteins of person sufferedwith liquefaction delay, Non-obstructive azoospermia and severe oligospermia thatmight lead to a better understanding of ongoing proteomics research of seminalplasma.MethodsThe research donors were recruited at the assisted reproductive center ofSouthern hospital, 29 seminal plasma samples were classified in accordance withWHO guidelines criteria and divided into four groups. Liquefaction delay group (7cases), Non-obstructive azoospermia group (NOA, 6 cases), severe oligospermiagroup (5 cases) and control group (11 cases). All the samples were detected bySELDI-TOF-MS (CM10 chip). After normalization of the data to total ion current,statistical analysis was performed to determine any significant differences in proteinexpression related to male infertility on this protein chip type.Results1. Liquefaction delay groupIn liquefaction group, 10 proteins were high expression than control group, themolecular weight were 8696.621 Da, 9770.076 Da, 9512.309 Da, 10202.64 Da,9617.759 Da, 10119.41 Da, 12345.16 Da, 12542.41 Da, 9407.785 Da, 16517.9 Darespectively (P＜0.05); 5 candidate proteins (8696.621 Da, 9770.076 Da, 9512.309Da, 10202.64 Da, 9617.759 Da )has significant difference, P＜0.01. At the same time, 9 proteins which have the character molecular weight2941.903 Da, 2669.629 Da, 2127.706 Da, 2922.53 Da, 2688.064 Da, 3454.267Da, 3602.682 Da, 7596.921 Da, 7630.573 Da were low expression than control group(P＜0.05), and the sole protein 2941.903 Da show significant difference. P＜0.01Screening from swiss-prot database and TrEMBL database of the protein whichhave significant difference between the two groups, it was indicated that Zinc fingerprotein, Dynein, light chain, Glutathione S-transferase isozyme and Cytochrome coxidase polypeptide maybe the suitable candicator of these proteins.2. Non-obstructive azoospermia groupComparing with control group, there were too much different proteinsexpressed in non-obstructive azoospermia group. Except 4 proteins have highexpress, others were decreased in non-obstructive azoospermia group, in which 4proteins have molecular weight character 7196.058 Da, 7630.573 Da, 7547.61 Da,7709.833 Da decreased in evidence. P＜0.01. Serine protease inhibitor Kazal-type 7precursor (MW: 7201.26) maybe the protein in significant difference according tothe swiss-prot database and TrEMBL database.3. Severe oligospermia groupScreening for specific proteins of oligospermia candidates, only two lowabundances proteins down-regulated expression in severe oligospermia group. Thespecific protein peaks possess of molecular weight 2806.517 Da, 15313.23 Da.4. Compare Severe oligospermia group with Non-obstructive azoospermia group15 different expression proteins were found between severe oligospermia groupand non-obstructive azoospermia group. Within them, 6 proteins were high expression in Non-obstructive azoospermia group, the mass weight were 10860.9Da, 5379.173 Da, 10202.64 Da, 10778.81 Da, 13788.28 Da, 13856.4 Da. (P＜0.05) 9 proteins were down-regulated in non-obstructive azoospermia group, themolecular weight were 2647.87 Da, 7196.058 Da, 7547.61 Da, 5780.493 Da,6393.921 Da, 7013.909 Da, 7059.844 Da, 7409.589 Da , 8621.5 Da (P＜0.05).Conclusion1. SELDI-TOF-MS offers the advantages of simple, rapid, high throughputexamination as well as high specificity and sensitivity, enables the comparativeanalysis of virtually any given protein-containing solution by using only minuteamount of clinical samples without destroying the proteins, is a suitable methodavailable for comparative proteomic research.2. In despite of the mechanisms of postejaculatory semen coagulation andliquefaction are, on the whole, understood. Apart from those macro molecularweight proteins, it has been found that many micro molecular weight proteinscontribute to this process. Zinc finger protein, Dynein, light chain, GlutathioneS-transferase isozyme and Cytochrome c oxidase polypeptide maybe thesuitable candicator of these proteins.3. Serine protease inhibitor maybe associated with NOA.Innovation point1. Screening difference proteins in seminal plasma of most conditions of maleinfertility by SELDI-TOF-MS.2. Discovering that many micro molecular weight proteins contribute to theprocess of liquefaction of seminal plasma for the first time. Zinc finger protein,Dynein, light chain, Glutathione S-transferase isozyme and Cytochrome c oxidase polypeptide maybe the suitable candidates of these proteins. As all ofthe proteins haven't mentioned in previous research, this report reinforce thetheory of liquefaction delayed.3. Report for the first time that the non-obstruction azoospermia patient hasmassive proteins difference expression with normal comparison group, Serineprotease inhibitor maybe one of them.4. Proposed for the first time that the azoospermia-correlation genes has targetorgan outside of testis. Abnormal gene expression maybe accompanied withprotein expression change in target organ. This caused the diseases which werediagnosed by molecular biology and pathology technology formerly simplifiesto diagnose by proteinchip directly.