Mass Spectral Analysis of Synthetic Peptides: Implications in Proteomics.

Sequence determination of peptides is a crucial step in mass spectrometry-based proteomics. Peptide sequences are determined either by database search or by sequencing using tandem mass spectrometry. Determination of all the theoretical expected peptide fragments and eliminating false discoveries remains a challenge in proteomics.

Detection of peptides with intact phosphate groups using MALDI TOF/TOF and comparison with the ESI-MS/MS.

A wide variety of post-translational modifications such as oxidation, phosphorylation, glycosylation, methylation, and acetylation play critical roles in cellular functions. Detection of post-translational modifications in proteins is important to understand their crucial roles in cellular functions. Identifying each modification requires special attention in mass spectral acquisition and analysis.

Inhibiting sperm pyruvate dehydrogenase complex and its E3 subunit, dihydrolipoamide dehydrogenase affects fertilization in Syrian hamsters.

Background/aims: The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium.

Methodology And Principal Findings: Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically.

Association of lactate, intracellular pH, and intracellular calcium during capacitation and acrosome reaction: contribution of hamster sperm dihydrolipoamide dehydrogenase, the E3 subunit of pyruvate dehydrogenase complex.

The role of dihydrolipoamide dehydrogenase (DLD), the E3 subunit of the pyruvate dehydrogenase complex (PDHc) in hamster sperm capacitation and acrosome reaction has been implicated previously. In this study, attempt has been made to understand DLD/PDHc involvement from the perspective of pyruvate/lactate metabolism. Inhibition of DLD was achieved by the use of a specific inhibitor, 5-methoxyindole-2-carboxylic acid.

Glucose-regulated protein precursor (GRP78) and tumor rejection antigen (GP96) are unique to hamster caput epididymal spermatozoa.

The immotile testicular mammalian spermatozoon gets transformed into a motile spermatozoon during 'epididymal maturation'. During this process, the spermatozoa transit from the caput to the cauda epididymis and undergo a number of distinct morphological, biophysical and biochemical changes, including changes in protein composition and protein modifications, which may be relevant to the acquisition of motility potential. The present proteome-based study of the hamster epididymal spermatozoa of caput and cauda led to the identification of 113 proteins spots using Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) analysis.

Proteomics-based study on asthenozoospermia: differential expression of proteasome alpha complex.

With a view to understand the molecular basis of sperm motility, we have tried to establish the human sperm proteome by two-dimensional PAGE MALDI MS/MS analysis. We report identification of 75 different proteins in the human spermatozoa. Comparative proteome analysis was carried out for asthenozoospermic and normozoospermic patients to understand the molecular basis of sperm motility.

The endothelial nitric oxide synthase Glu298Asp polymorphism is not a risk factor for endometriosis in south Indian women.

Objective: To investigate whether the eNOS gene influences the risk of developing endometriosis in south Indian women.

Study Design: The single nucleotide polymorphism, Glu298Asp, in exon7 of the eNOS gene was tested for association in a case-control study of 232 affected women and 210 women with no evidence of disease. All the women were infertile, ascertained from the same infertility clinic.

Inhibition of in vitro capacitation of hamster spermatozoa by nitric oxide synthase inhibitors.

In an attempt to understand the role of nitric oxide(NO) in sperm capacitation, in the present study, hamster spermatozoa were used to evaluate the effects of NO on motility, viability, hyperactivation, capacitation and protein tyrosine and serine phosphorylation using specific inhibitors of nitric oxide synthase (NOS); namely L-NAME (N-nito-L-aginine methyl ester) and 7-Ni (7-nitroindazole). The results indicated that L-NAME inhibits sperm motility, hyperactivation and acrosome reaction where as 7-Ni inhibits only hyperactivation and acrosome reaction thus implying that NOS inhibitors exhibit subtle differences with respect to their effects on sperm functions. This study also provides evidence that NOS inhibitors inhibit sperm capacitation by their ability to modulate protein tyrosine phosphorylation.

Role of signaling pathways in regulating the capacitation of mammalian spermatozoa.

Mammalian testicular spermatozoa are immotile and incompetent for fertilization. They acquire motility during epididymal maturation and fertilizing ability during a second phase of maturation in the female reproductive tract, termed as capacitation. Capacitation was discovered independently by Austin and Cang in early 1950s and was defined as the obligate period of residency of spermatozoa in the female reproductive tract, which confers on the spermatozoa the ability to fertilize an oocyte.

The differential effects of guanosine tetraphosphate on open complex formation at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1.

The effects of guanosine tetraphosphate (ppGpp) on inhibition of single-round in vitro transcription and on the kinetics of open complex formation were investigated at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1. The two promoters differ in their saturation characteristics and sensitivities to ppGpp. With a 10:1 molar ratio of RNA polymerase (RNAP) to DNA, saturation of transcription activity and weak inhibition (approximately 30%) are observed at rplJ, in contrast to the weak activity and strong inhibition (approximately 80%) at rpsA P1.