Kaleem Ullah

Switchable boronate affinity materials for thermally modulated capture, separation and enrichment of cis-diol biomolecules

A new thermoresponsive boronate affinity chromatographic material, P(N-isopropylacrylamide-4-vinylphenylboronic acid) P(NIPAAm-co-VPBA) grafted silica featuring a thermally switchable property, was prepared and successfully employed for the specific capture–release of cis-diol biomolecules with simpler adjustment to the temperature instead of the conventional need to change the pH of the mobile phase.

Study of rat hypothalamic proteome by HPLC/ESI ion trap and HPLC/ESI-Q-TOF MS.

The proteomic profile of hypothalamus, a key organ of CNS, is explored here by employing two widely used MS techniques, i.e. HPLC/ESI‐ion trap and HPLC/ESI‐quadrupole‐TOF MS. Strong cation exchange is used for the fractionation of peptides and protein search engine MASCOT is employed for data query. One hundred and thirty six proteins with 10 973 peptides were identified by HPLC/ESI‐ion trap MS, while 140 proteins with 32 183 peptides were characterized by HPLC/ESI‐quadrupole‐TOF MS. Among the total 198 proteins identified in both experiments, 78 proteins were common in both sets of conditions. The rest of the 120 proteins were identified distinctly in both MS strategies, i.e. 58 unique proteins were found using the quadrupole‐TOF while 62 were found with the HPLC/ESI‐ion trap. Moreover, these proteins were classified into groups based on their functions performed in the body. Results presented here identified some important signal and cellular defense proteins inevitable for survival in stressed conditions. Additionally, it is also shown that any single MS strategy is not reliable for good results due to loss of data depending on sensitivity of the instrument used.

Distortion of homeostatic signaling proteins by simulated microgravity in rat hypothalamus: A16O/18O‐labeled comparative integrated proteomic approach

Microgravity generates oxidative stress in central nervous system, causing distortion of various vital signaling cascades involved in many homeostatic functions. Here, we performed comparative 16O/18O labeled integrated proteomic strategy to observe the differential expression of signaling proteins involved in homeostasis. In this study, rat‐tail suspension model is employed to induce simulated microgravity in CNS. By wide proteomic analysis, total of 35 and 97 significantly differentially expressed proteins were found by HPLC/ESI‐TOF and HPLC‐Q‐TOF analysis, respectively. Among the total of 132 proteins quantified, 25 proteins were found related to various signaling cascades. Protein Thy‐1, 14‐3‐3 gamma, 14‐3‐3 epsilon, 14‐3‐3 theta, 14‐3‐3 eta, and 14‐3‐3 beta/alpha proteins, calmodulin and calcium/calmodulin‐dependent protein kinase type‐II subunit beta were found upregulated under the influence of simulated microgravity. These proteins are found involved in disrupting homeostatic pathways like sleep/wake cycle, drinking behavior, hypothalamic‐pituitary‐adrenocortical regulation and fight and/or flee actions under stress. Furthermore, MS results for protein Thy‐1 were verified by Western blot analysis showing the quantification accuracy of MS instruments. Results presented here will serve as means to understand the mechanism of action of microgravity and further reference for future detailed study of consequences of microgravity on astronauts and their possible countermeasures.

A newly discovered neurotoxin ADTIQ associated with hyperglycemia and Parkinson's disease.

BACKGROUND Diabetes is associated with an increased risk of Parkinsons disease (PD). Number of studies have suggested that methylglyoxal (MGO) induced by diabetes is related to PD. However, very little is known about its molecular mechanism. On other hand, 1-acetyl-6, 7- dihydroxyl-1, 2, 3, 4- Tetrahydroisoquinoline(ADTIQ) is a dopamine (DA)-derived tetrahydroisoquinoline (TIQ), a novel endogenous neurotoxins, which was first discovered in frozen Parkinsons disease human brain tissue. While ADTIQ precursor methylglyoxal was also found in diabetic patients related to the glucose metabolism and diabetic patients. METHODS LC-MS/MS, 1H NMR and infrared spectroscopy identified the structure of ADTIQ. The Annexin V-FITC/PI, MTT and western blot analysis were used to measure the neurotoxicity of ADTIQ. The levels of ADTIQ and methylglyoxal were detected by LC-MS/MS. RESULTS Here we report the chemical synthesis of ADTIQ, demonstrate its biosynthesis in SH-SY5Y neuroblastoma cell line and investigate its role in the pathogenesis of PD. In addition, a significant increase in the level of ADTIQ was detected in the brains of transgenic mice expressing mutant forms (A53T or A30P) of α-synuclein. ADTIQ also reduced the cell viability and induced mitochondrial apoptosis in dopaminergic cells, suggesting that ADTIQ acts as an endogenous neurotoxin and potentially involved in the pathogenesis of PD. Methylglyoxal, a major byproduct of glucose metabolism and abnormalities in glucose metabolism could influence the levels of ADTIQ. Consistent with the hypothesis, increased levels of ADTIQ and methylglyoxal were detected in the striatum of diabetic rats and SH-SY5Y cells cultured in the presence of high glucose concentrations. CONCLUSIONS Increased levels of ADTIQ could be related with Hyperglycemia and death of dopaminergic neurons. GENERAL SIGNIFICANCE The increased levels of ADTIQ could be a reason of dopamine neuron dysfunction in diabetes. Therefore, ADTIQ may play a key role in increasing the risk for PD in patients with diabetes.

Separation of peptides with an aqueous mobile phase by temperature‐responsive chromatographic column

Peptide separation technology is significant and is still an analytical challenge in proteomic studies. We report a simple preparation method for poly(N-isopropylacrylamide) grafted silica through the copolymerization of N-isopropylacrylamide with acetyl moieties immobilized on the silica surfaces. Differential scanning calorimetry results indicated that the prepared silica exhibited a sharp phase transition at 35.03°C. Silica grafted with poly(N-isopropylacrylamide) was evaluated as a temperature-responsive chromatography medium for the separation of peptides using 0.2 M NaCl solution as a mobile phase. Results indicated that at 10°C, the peptides were not resolved, but baseline separation with prolonged retention time at 50°C was attained. Particularly, a mixture of four peptides was efficiently separated within 8 min. The theoretical plate number of every peptide was more than 2500, and the resolutions were more than 3.40. The increased selectivity of the temperature-responsive column resulted from the temperature-modulated hydrophobic interaction with peptides. The retention times of these peptides were related to their hydrophobicities. This protocol provided a reliable set of chromatographic tool usable across all research and development applications that required isolation and analysis of peptides. It may represent a step forward in the complex analysis of hydrophobic and other proteins.

Methylglyoxal increases dopamine level and leads to oxidative stress in SH-SY5Y cells

More and more studies have suggested that methylglyoxal (MGO) induced by type-2 diabetes is related to Parkinsons disease (PD). However, little is known about the molecular mechanism. In this study, we explored the MGO toxicity in neuroblastoma SH-SY5Y cells. Neurotoxicity of MGO was measured by mitochondrial membrane potential, malondialdehyde, and methylthiazoletetrazolium assays. The levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) were detected by liquid chromatography-mass spectrometry/mass spectrometry. The expressions of tyrosine hydroxylase (TH) and dopamine transporter (DAT) were detected by reverse transcriptase polymerase chain reaction and western blot analysis. The results showed that MGO induced an increase in TH and DAT expressions in SH-SY5Y neuroblastoma cells, while the levels of dopamine, DOPAC, and endogenous neurotoxin salsolinol also increased. Aminoguanidine (AG) is an inhibitor of MGO. It was found that AG could decrease the reactive oxygen species (ROS) level induced by MGO, but could not inhibit an increase of TH, DAT and dopamine. The increase of dopamine, DOPAC and salsolinol levels could lead to high ROS and mitochondrial damage. This study suggests that ROS caused by dopamine could contribute to the damage of dopaminergic neurons when MGO is increased during the course of diabetes.

Arterial vascular cell line expressing SSAO: a new tool to study the pathophysiology of vascular amine oxidases

Semicarbazide-sensitive amine oxidase (SSAO) widely exists in nature, mainly expressed at significant levels in vasculature. It plays a detrimental role in vascular diseases, particularly atherosclerosis, which occurs mainly in arteries. Herein we for the first time present SSAO expression in arterial lineage of vascular cell line, i.e., human umbilical arterial endothelial cell (HUAEC). Firstly, two commercially available gene transfection reagents were compared to determine high transfection efficiency and then the expression behavior of HUAEC:SSAO was characterized. Furthermore, our model was also been compared with commonly used human embryonic kidney (HEK) cell transfected with the same vector. For enzymatic assay, an in-house developed highly sensitive high performance liquid chromatography electron spray ionization mass spectrometry method was applied. Results indicated that the maximal transfection efficiency in HUAEC was detected by JetPEI™ and transfected protein was expressed at membrane and cytosol of different clones. No significant variations were observed in HUAEC between cell passages 1 and 7, although HEK cell displayed twofold higher SSAO expression level than HUAEC. The transfected SSAO was shown to be released into the cell-culture medium. Both cellular and released types of SSAO exhibited monomer and dimer structural forms. The cytotoxicity determination exhibited large number of viable cells after transfection with JetPEI™. Differential expression characterization of this new cell line demonstrates the correct behavior of SSAO in arterial endothelial cells and also provides a real physiological environment to elucidate the unclear role of this enzyme. In addition, our cellular model could partly solve the problems raised by the loss of enzyme expression found in cultured endothelial cells. This model could also be a useful tool for proteomic base study, screening of interacting protein and analysis of compounds that could modify its activity for therapeutic purposes.

Determination of Endogenous Neurotoxin 1-Acetyl-6,7-Dihydroxyl-1,2,3,4-Tetrahydroisoquinoline in Rat Substantia Nigra by High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

The endogenous neurotoxin, 1-acetyl-6,7-dihydroxyl-1,2,3,4 –tetrahydro isoquinoline (ADTIQ), is acatecholamine isoquinoline, indicating its possible involvement in Parkinsons disease. Moreover, it has been assumed to be a bridge between diabetes and Parkinsons disease because metabolic synthesis resembles glucose metabolism. Therefore, the accurate determination of ADTIQ could play an important role in diagnosis of diabetes and Parkinsons disease. Herein, a sensitive and precise method was established to determine ADTIQ by high performance liquid chromatography-electrospray ionization-tandem mass spectrometry in multiple reaction monitoring mode. The [M+H]+ precursor product ions for ADTIQ were 208.1/190.1 and linear range of calibration curve was from 1.0 to 250.0 nM. The limit of detection and limit of quantification of the developed method were 0.2 nM and 1 nM, respectively. The inter-day, intra-day precision, accuracy, and recoveries were acceptable for biological samples. Furthermore, the reported method was used to determine ADTIQ levels in the substantia nigra of rats. The results indicated that the developed method was highly sensitive and accurate for the determination of ADTIQ.

Differential expression of semicrabazide-sensitive amine oxidase (SSAO) in human umbilical arterial tissue, E.coli BL21, HEK and HUAEC

Semicarbazide-sensitive amine oxidase (SSAO) is multifunctional enzyme which is highly express in vasculature, smooth muscle and adipose tissues. Clinical and experimental studies suggest that increased activity and the catalytic product of deamination may be toxic and cause the tissue damage. Correlation between the SSAO activity and severity of different serious pathological condition such as Diabetes, Alzheimers disease and atherosclerosis have been widely reported but pathophysiological meaning of this enzyme is not yet discovered. Therefore, research related to SSAO as therapeutic objective are becoming more important. We present liquid chromatography electrospray ionization mass spectrometry (LC-MS/MS) based comparative enzymatic activity and expression study in human umbilical tissue, universal recombinant expression system E.coli; commonly used cell line i.e., human embryonic kidney cell (HEK) and naturally SSAO expressive primary vascular cell line i.e., human umbilical arterial endothelial cell (HUAEC). Our results indicate that HEK:SSAO is best model among these while the second most abundant activity was observed in E.coli BL21:SSAO, with a rough estimation HEK:SSAO shows two to three fold higher activity than HUAEC. The concept of comparison provides new insight on this enzyme expression. Our models also open new possibilities for the evaluation of potential compounds that could modify SSAO activity for therapeutic determinations.