Dodecyl Sulfate Electrophoresis Research Articles

Salivary Protein and Electrolyte Profiles during Primary Teeth Eruption: A Cross-Sectional Study.

This study aimed to assess the qualitative changes in the saliva during the process of primary teeth eruption. This cross-sectional study was conducted on 147 children from 2 to 48 months, of which 49 were in group A (no erupted primary teeth), 53 were in group B (at least one active erupting primary tooth), and 45 were in group C (eruption of all 20 primary teeth was completed). Salivary proteins were evaluated by sodium dodecyl sulfate electrophoresis with polyacrylamide gel, while the concentrations of salivary sodium, potassium, chloride, and calcium ions were evaluated by ion selective electrodes. The data were analyzed using ANOVA and Bonferroni tests (alpha = 0.05). The concentration of proteins with molecular weights of 20-30 KDa was significantly higher in group A, and it gradually decreased with age. The concentration of proteins with molecular weights of 50-60 KDa in group B was significantly lower than those of groups A and C. The calcium ion concentration in group A was significantly higher than that of the other groups. The concentration of potassium ions was minimal in group C. The proteins and electrolyte profiles of the subjects' saliva changed in the process of primary tooth eruption. The highest concentrations of proteins such as statherin, histatin, P-B peptide, and cystatin and the lowest concentrations of proteins such as amylase were present in group B.

Evaluating the effect of cooking temperature and time on collagen characteristics and the texture of hog maw.

This study evaluated the texture of hog maw and the degradation of Types I and III collagen in the intramuscular connective tissue (IMCT) of hog maw at different cooking temperatures (75-95°C) and times (50-130 min). The cooking loss, shear force, collagen content, collagen solubility, and IMCT strength of hog maw cooked in water baths were measured. The instrumental texture profile analysis showed that the brittleness, springiness, chewiness and hardness of the cooked hog maw significantly increased with the increase of cooking temperature, while the hardness, springiness and chewiness increased first and then decreased with increasing cooking time. Cooking loss exhibited a 38% increase between the raw meat and meat cooked at 95°C. The collagen solubility significantly increased from 5.5 mg/g for raw meat to 8.6 mg/g for meat cooked at 95°C, accompanied by decreases in the shear force and IMCT strength associated with the increase in cooking temperature and time. These results show that the texture and collagen characteristics of hog maw are dramatically affected by the cooking temperature and time. Sodium dodecyl sulfate electrophoresis and immunofluorescence staining further showed that collagen degradation occurred after cooking, and the degradation of Type I collagen was higher than that of Type III collagen. These results indicated that the degradation of Type I collagen was mainly responsible for the sensory and textural improvements of the cooked hog maw.

SEED PROTEINS ANALYSIS OF FESTUCA SERIES PSAMMOPHILAE M. PAWLUS AND OVINAE M. PAWLUS (POACEAE)

According to the type of leaf anatomy, narrow-leaved fescues could be divided to three main groups: species with leaves as in F estuca rubra (sclerenchyma strands small, numerous), type F. valesiaca (3 main and big sclerenchyma strands + adventive) and type F. ovina (sclerenchyma ring). The species of the last group were chosen in our study. The seed proteins variation in natural populations of Festuca vaginata , F. psammophila , F. pallens , F. polesica and F. ovina from Ukraine and Bulgaria was examined by means of polyacrylamide (PAGE) and sodium dodecyl sulphate (SDS-PAGE) electrophoresis. These fescues exhibit high variability and overlap of morphological and anatomical characters, including diagnostic ones, leading to identification difficulties and uncertainties in their taxonomy. The purpose of the present study was to analyze seed protein composition in order to reveal systematic relationships among the above mentioned taxa of genus Festuca . The results showed that F. vaginata , F. psammophila and F. polesica are closely related. The species F. pallens was relatively distant from the above mentioned three species. The specific position of F. pallens is confirmed also by its ecological characteristics– it is the only species growing on carbonate rocks, while the rest of taxa are typical psammophytes. The results showed distinct differences between Ukrainian populations of F. p allens and F. psammophila and confirmed occurrence of latter in Ukrainian Roztocha. The cluster patterns were somewhat inconsistent in regard to different types of proteins (PAGE, SDS-PAGE). The only exception is F. ovina , which always occupies a separate position. This confirms its remote position within the studied group of the genus Festuca and supports the view to consider F. ovina s.str. as a separate series Ovinae among the species with sclerenchyma ring. Whereas all other studied species ( F . vaginata , F. psammophila , F. pallens , F. polesica ) should be considered as a separate series of closely related taxa – series Psammophilae .

Expression, purification and characterization of cellobiose dehydrogenase mutants from Phanerochaete chrysosporium in Pichia pastoris KM71H strain

Production of soluble cellobiose dehydrogenase (CDH) mutant proteins previously evolved on the surface of S. cerevisiae yeast cells was established for use in biosensors and biofuel cells. For this purpose, mutant cdh genes tm (D20N, A64T, V592M), H5 (D20N, V22A, A64T, V592M) and H9 (D20N, A64T, T84A, A261P, V592M, E674G, N715S) were cloned to pPICZ? plasmid and transformed into Pichia pastoris KM71H strain for high expression in a soluble form and kinetic characterization. After 6 days of expression under methanol induction, the CDHs were purified by ultrafiltration, ion- -exchange chromatography and gel filtration. Sodium dodecyl sulfate electrophoresis confirmed the purity and presence of a single protein band at a molecular weight of 100 kDa. Kinetic characterization showed that the H5 mutant had the highest catalytic constant of 43.5 s-1 for lactose, while the mutant H9 showed the highest specificity constant for lactose of 132 mM-1 s-1. All three mutant proteins did not change the pH optimum that was between 4.5 and 5.5. Compared to the previously obtained wild types and mutants of CDH from Phanerochaete chrysosporium, the variants reported in this article had higher activity and specificity that together with high protein expression rate in P. pastoris, makes them good candidates for use in biotechnology for lactobionic acid production and biosensor manufacture.

Glutathione S-Transferase: Purification and Characterization of from Cherry Laurel (Prunus laurocerasus L.) and the Investigation In Vitro Effects of Some Metal Ions and Organic Compounds on Enzyme Activity

In this study, the cherry laurel flesh fruit was purified to obtain glutathione S-transferase, a well-known antioxidant enzyme. Enzyme purification was performed using two separate processes: gel filtration and affinity chromatography. The sodium dodecyl sulfate electrophoresis method was used for determining enzyme purity. The optimum pH, optimum temperature, optimum ionic strength, stable pH, and KM and Vmax values for glutathione and 1-chloro-2,4-dinitrobenzene were obtained for the enzyme as follows: 7.0 in the K-phosphate buffer, 30 °C, 0.125 M, 6.5 in the K-phosphate buffer, 0.344 mM, 0.89 mM, and 0.098 EU/ml, 0.214 EU/ml. Lineweaver-Burk graphs were used to examine the in vitro enzyme activity in order to determine the inhibitory effects of some metal ions such as Cd2+, Ni2+, Cu2+, and Mg2+ and organic compounds such as sodium dodecyl sulfate (SDS), ascorbic acid (vitamin C), benzoic acid, and ethylenediaminetetraacetic acid (EDTA). The IC50 and Ki values for each of the metal ions and organic molecules were calculated. According to the results, Ca2+ metal ion and sodium dodecyl sulfate compounds were found to be the best inhibitor with a Ki value of 0.06 ± 0.04 and 0.20 ± 0.27, respectively.

Optimization of the cydex blue assay: A one-step colorimetric protein assay using cyclodextrins and compatible with detergents and reducers.

Sodium dodecyl sulfate electrophoresis (SDS) is a protein separation technique widely used, for example, prior to immunoblotting. Samples are usually prepared in a buffer containing both high concentrations of reducers and high concentrations of SDS. This conjunction renders the samples incompatible with common protein assays. By chelating the SDS, cyclodextrins make the use of simple, dye-based colorimetric assays possible. In this paper, we describe the optimization of the assay, focussing on the cyclodextrin/SDS ratio and the use of commercial assay reagents. The adaptation of the assay to a microplate format and using other detergent-containing conventional extraction buffers is also described.

Amyloid fibrillogenesis of lysozyme is suppressed by a food additive brilliant blue FCF

At least 30 different human proteins can fold abnormally to form the amyloid deposits that are associated with a number of degenerative diseases. The research presented here aimed at understanding the inhibitory potency of a food additive, brilliant blue FCF (BBF), on the amyloid fibril formation of lysozyme. Our results demonstrated that BBF was able to suppress the formation of lysozyme fibrils in a dose-dependent fashion. In addition, the structural features and conformational changes in the lysozyme samples upon the addition of BBF were further characterized using circular dichroism spectroscopy, nile red fluorescence spectroscopy, turbidity assay, and sodium dodecyl sulfate electrophoresis. Through molecular docking and molecular dynamics simulations, BBF’s mechanism of action in lysozyme fibrillogenesis inhibition was found to be initiated by binding with the aggregation-prone region of the lysozyme. We believe the results from this research may contribute to the development of effective therapeutics for amyloidoses.

Urinary gamma-glutamyl transferase (GGT) as a marker of tubular proteinuria in dogs with canine leishmaniasis, using sodium dodecylsulphate (SDS) electrophoresis as a reference method

In order to assess if urinary γ- glutamyl transferase (GGT) identify tubular proteinuria in leishmaniotic dogs, the GGT/urinary creatinine (UC) ratio was calculated in 39 leishmaniotic dogs. According to sodium dodecylsulphate–agarose gel electrophoresis, the dogs had albuminuria (A, n = 10), glomerular (G, n = 3), tubular (T, n = 4) or mixed proteinuria (M, n = 22). The median GGT/UC ratio was 0.3, 0.3, 2.2, and 7.5, in groups G, A, M, and T, respectively. Statistically significant differences were found between groups G and M (P = 0.002), G and T (P < 0.001), A and M (P < 0.001), and A and T (P < 0.001). Median values were higher in dogs with tubular components of proteinuria (M/T, 2.5) than in dogs without tubular components of proteinuria (A/G, 0.3), and in dogs with tubular proteinuria (T, 7.5) than in dogs with non-tubular proteinuria (NT, 1.0). GGT/UC values >0.81 or >2.64 could identify dogs in the M/T or T groups, respectively. Therefore, GGT/UC might be useful for the management of leishmaniotic dogs.

Detection of novel auto-antigens in patients with recurrent miscarriage: description of an approach and preliminary findings

AimTo develop and test a protocol for isolation of potential auto-antigens from chorionic tissue that may be linked to recurrent miscarriage (RM).MethodsThe strategy included: 1) isolation of IgGs tightly bound to chorionic tissue of RM patients by protein G chromatography; 2) construction of affinity columns using the chorionic antibodies for isolation of auto-antigens; 3) enrichment of auto-antigens from detergent extracted solution of chorionic proteins by affinity chromatography; 4) separation by dodecyl sulfate-electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry identification.ResultsFive potential auto-antigens were detected: neutral alpha-glucosidase AB, endoplasmin, transitional endoplasmic reticulum ATPase, putative endoplasmin-like protein, and cytoplasmic actin 2.ConclusionsWe developed a strategy for identification of auto-antigens in the chorionic tissue of women with RM, which could be of diagnostic and prognostic value.

Allelic diversity of high molecular weight glutenin subunits (HMW-GS) in Iranian Aegilops tauschii Coss. accessions by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

Variation of high molecular weight glutenin subunits (HMW-GS) in 28 Iranian Aegilops tauschii (2n = 2x = 14, DD) accessions studied by sodium dodecyl sulphate electrophoresis method (SDS-PAGE). The results showed high variation of HMW-GS in the accessions. The range of frequency in 14 HMW-GS combinations was 3.57–25 % in the accessions. AMOVA showed the molecular variance between the geographic areas was lower than within the geographic areas. According to Nei’s genetic diversity, the highest diversity levels were in Semnan, Golestan and Azarbayjan, on the other hand the lowest levels of diversity were found in Khorasan, Gilan and Mazandaran accessions. Hence, the Caspian Sea South East accessions also Azerbayjan in Iran have more diversity. AMOVA did not show variance between strangulata and tauschii but there was more genetic diversity in ssp. tauschii subspecies in comparison of ssp. strangulata according to Nei’s gene diversity and Shannon information index. It showed Iranian Ae. tauschii have a good potential for bread making quality improvement in bread wheat.

Identification of differentiation-related proteins in gastric adenocarcinoma by comparative proteomics

Objective To identify differentiation-related proteins in gastric adenocarcinoma by comparative proteomics.Methods The holoproteins of human gastric adenocarcinoma tissues in 4 well differentiated and 4 poorly differentiated were measured by sodium dodecyl-sulphate (SDS) electrophoresis and liquid chromatography-mass spectrometry (LC-MS),and differentiation-related proteins in gastric adenocarcinoma were analyzed.Some proteins obtained through proteomics were tested by Real-time quantitative polymerase chain reaction detecting system (QPCR) and Western blotting in the tissues of gastric carcinoma.Results Fifteen differential protein bands were found in gastric adenocarcinoma,among them 355 proteins were identified by LC-MS.The proteins such as high abundant proteins and structural proteins were ruled out,then 8 proteins were selected and these may be meaningful,which included cofilin,proteasome activator subunitl,ras-related protein rab-7a,filamin a,pyruvate kinase,glutathione s-transferasesπ-1,peroxiredoxin 5,actin-related protein.These proteins are involved in the signal transduction,maintenance of cell structure,and regulate cell proliferation and apoptosis.The expression of 3 proteins identified by Western blotting and QPCR was consistent with the results obtained by proteomics.Conclusion Differential proteins are found in gastric adenocarcinoma tissues with different differentiation degrees. Key words: Gastric neoplasms; Comparative proteomics; Cell differentiation

Purification of Soluble Acetylcholinesterase from Sheep Liver by Affinity Chromatography

The purpose of this study was to develop a protocol for the purification of acetylcholinesterase (AChE, acetylcholine acetylhydrolase, E.C.3.1.1.7) enzyme and to extend a purification method for further enzyme characterization. A further aim was to study whether the edrophonium's pharmacologic action is due primarily to the inhibition or inactivation of AChE at sites of cholinergic transmission. The purification of a soluble AChE from sheep liver using affinity chromatography on Concanavalin A-Sepharose 4B and edrophonium-Sepharose 6B is studied. The affinity matrix was synthesized by coupling an inhibitor edrophonium to epoxy-activated Sepharose at flow rate of 0.5ml/min. AChE is a pivotal enzyme in the cholinergic nervous system. Its primary function is to catalyze hydrolysis of released acetylcholine (ACh) and thus maintain homeostasis of this neurotransmitter in the central and peripheral nervous systems. Hence, AChE is important in both pharmacological and toxicological mechanisms. It was purified 842-fold with a specific activity of 21U/mg protein. Sodium dodecyl sulfate (SDS) electrophoresis resulted in a monomeric molecular weight of 67.04kDa, while on gel chromatography using Sephacryl S-200 under nondenaturing conditions to be 201.5kDa. Based on the molecular weight obtained by gel filtration, the purified AChE was assumed to be a tetrameric form.

Recent advances in electrophoretic techniques for the characterization of protein biomolecules: A poker of aces

The four classical modes of electrophoresis of protein molecules (sodium dodecyl sulphate electrophoresis, SDS-PAGE, isoelectric focusing, IEF, and immobilized pH gradients, IPGs, two-dimensional maps, 2D, and capillary electrophoresis, CE) are here reviewed, with special emphasis on recent innovations. Thus, in the case of SDS-PAGE, a novel method, consisting in focusing SDS-protein micelles against a gradient of cationic charges grafted onto a polyacrylamide gel is presented. In the case of IEF, the recent decoding of the structure, polydispersity, molecular mass distribution and buffering properties of the soluble carrier ampholyte buffers are here discussed. In regard to two dimensional mapping, recent instrumentation for performing 2D maps in horizontal, large gel slabs (up to 30 cm × 40 cm) and in a radial format for the SDS dimension is here evaluated. Finally, in the case of CE, three major applications are presented: a thorough study of capillary IEF and of all experimental variables, a method of importance in screening of rDNA products; the possibility of running proteins and peptide separations in very acidic, amphoteric, isoelectric buffers in absence of any capillary coating; finally, the possibility of producing a facile, user friendly, covalent coating of the wall silanols via bonding of quaternarized piperazines endowed with an iodinated tail. In acidic, volatile buffers, such protein/peptide runs can be directly interfaced with mass spectrometry instrumentation.

Clinical features of severe acquired ADAMTS13 deficiency in thrombotic thrombocytopenic purpura: the Korean TTP registry experience

The clinical significance of ADAMTS13 activity for response to treatment, mortality rate, recurrence, and prognosis is unclear. Therefore, we investigated the characteristics of severe ADAMTS13 deficiency and evaluated its prognostic features in Thrombotic thrombocytopenic purpura (TTP). The Korean TTP Registry includes 66 patients from 13 teaching hospitals in Korea who received the diagnosis of TTP from January 2005 to December 2008. Blood samples obtained upon admission were sent for ADAMTS13 analysis (multimer analysis by sodium dodecyl sulfate electrophoresis) to a central laboratory along with patient clinical information. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels (P=0.001) than patients with non-severe ADAMTS13 deficiency. Although severe ADAMTS13 deficiency was associated with better response rate (75 vs. 53%, P=0.145), remission rate (81 vs. 61%, P=0.209), and mortality rate (19 vs. 31%, P=0.508) than non-severe ADAMTS13 deficiency, treatment outcomes did not differ significantly between groups. After adjusting for clinical and laboratory features, multivariate analysis did not reveal any independent risk factors for TTP-associated mortality. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels at presentation, but severe ADAMTS13 activity deficiency at TTP diagnosis does not appear to have prognostic significance.

Clinical Characteristics of Thrombotic Thrombocytopenic Purpura with Severe ADAMTS13 Deficiency

AbstractAbstract 4666The clinical significance of ADAMTS13 activity for response to treatment, mortality rate, recurrence, and prognosis is unclear. Therefore, we investigated the characteristics of severe ADAMTS13 deficiency and evaluated its clinical significance in Thrombotic thrombocytopenic purpura (TTP). The Korean TTP Registry includes 66 patients from 13 teaching hospitals in Korea who received the diagnosis of TTP from January 2005 to December 2008. Blood samples obtained upon admission were sent for ADAMTS13 analysis (multimer analysis by sodium dodecyl sulfate electrophoresis and/or ELISA) to a central laboratory along with patient clinical information. After 6 months, patient data regarding treatment, response, and prognosis were collected on standardized report forms. Patients with severe ADAMTS13 deficiency had lower serum creatinine levels (P=0.001) and WBC counts (P=0.050) than patients with non-severe ADAMTS13 deficiency. Although severe ADAMTS13 deficiency was associated with better response rate (75% vs 53%, P=0.145), remission rate (81% vs 61%, P=0.209), and mortality rate (19% vs 31%, P=0.508) than non-severe ADAMTS13 deficiency, treatment outcomes did not differ significantly between groups. After adjusting for clinical and laboratory features, multivariate analysis did not reveal any independent risk factors for TTP-associated mortality. Patients with severe ADAMTS13 deficient had lower serum creatinine levels and WBC counts at presentation but Severe ADAMTS13 activity deficiency at TTP diagnosis does not appear to have prognostic significance. Disclosures:No relevant conflicts of interest to declare.

Simultaneous Presence of PrtH and PrtH2 Proteinases in Lactobacillus helveticus Strains Improves Breakdown of the Pure α s1 -Casein

Lactobacillus helveticus can possess one or two cell envelope proteinases (CEPs), called PrtH2 and PrtH. The aim of this work was to explore the diversity of 15 strains of L. helveticus, isolated from various origins, in terms of their proteolytic activities and specificities on pure caseins or on milk casein micelles. CEP activity differed 14-fold when the strains were assayed on a synthetic substrate, but no significant differences were detected between strains possessing one or two CEPs. No correlation was observed between the proteolytic activities of the strains and their rates of acidification in milk. The kinetics of hydrolysis of purified α(s1)- and β-casein by L. helveticus whole cells was monitored using Tris-Tricine sodium dodecyl sulfate (SDS) electrophoresis, and for four strains, the peptides released were identified using mass spectrometry. While rapid hydrolysis of pure β-casein was observed for all strains, the hydrolysis kinetics of α(s1)-casein was the only criterion capable of distinguishing between the strains based on the number of CEPs. Fifty-four to 74 peptides were identified for each strain. When only PrtH2 was present, 22 to 30% of the peptides originated from α(s1)-casein. The percentage increased to 41 to 49% for strains in which both CEPs were expressed. The peptide size ranged from 6 to 33 amino acids, revealing a broad range of cleavage specificities, involving all classes of amino acids (Leu, Val, Ala, Ile, Glu, Gln, Lys, Arg, Met, and Pro). Regions resistant to proteolysis were identified in both caseins. When strains were grown in milk, a drastic reduction in the number of peptides was observed, reflecting changes in accessibility and/or peptide assimilation during growth.

Deletion of a terminal residue disrupts oligomerization of a transmembrane α-helixThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular &amp; Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

In studies of the structural biology of membrane proteins, the success of strategies based on the "divide and conquer" approach, where peptides are used to model the individual transmembrane (TM) alpha-helices of membrane proteins, depends on the correct identification of the membrane-embedded TM alpha-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of a Lys-tagged TM2 alpha-helix of myelin proteolipid protein (PLP), of parent sequence KKKK-66AFQYVIYGTASFFFLYGALLLAEG89-KKKK along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and of a hydrophobic mutant Leu-90. Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS), we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is enough to change its intrinsic properties, with TM2 66-89 displaying only a monomeric form, but with dimers arising for the other 3 peptides. A novel use of trifluoroethanol (TFE) as a maximal helix-supporting solvent demonstrated that peptides containing residues at positions 90 and (or) 90-91 displayed significantly increased helical content vs. the TM2 parent peptide. The findings suggest that deletion of critical C-terminal residue(s) tends to reposition the helix terminus toward the membrane-aqueous interface. Our overall results emphasize the potential influence of boundary residues on TM properties when using peptides as models for TM alpha-helices, and may implicate a role for these residues in membrane protein folding and assembly.

Electrophoresis today and tomorrow: Helping biologists' dreams come true

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two-dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High-throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser-induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH-driven mobilization, provides efficient separations and on-line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single-cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single-molecule detection.

Analysis of monoclonal antibody product heterogeneity resulting from alternate cleavage sites of signal peptide

Signal peptides used in biosynthesis of proteins are cleaved at a very specific site by signal peptidase during posttranslational translocation of cytoplasmic proteins across the membrane. In some cases, however, there can be cleavage at nonspecific sites, giving rise to heterogeneity in the mature protein, which manifests itself as either elongation or truncation of the N terminus of the mature protein. When used as biopharmaceutical therapeutics, such heterogeneities may be a cause for concern, depending on the nature of the heterogeneity. This article describes the determination of such heterogeneity by peptide mapping in both the heavy chain and the light chain (LC) of a Chinese hamster ovary (CHO) cell-expressed monoclonal antibody (mAb). The peptide map method described here was capable of detecting the extended N-terminal peptides at levels as low as 1% relative to the peak area of the intact N-terminal peptide. The LC of a mAb product was truncated at its N termini by two amino acid residues at approximately 3–4% levels, resulting from alternate signal peptide cleavage. This article describes the quantitation of this truncation by liquid chromatography–mass spectrometry (LC–MS) peptide mapping. Also described is analysis and characterization of LC truncation by reduced and denatured capillary electrophoresis in sodium dodecyl sulfate (CE–SDS). The truncated mAb, which was devoid of the two N-terminal amino acids, was engineered and shown to migrate as the “pre-LC” peak in reduced CE–SDS assay. The amount of the pre-LC peak recovered from the CE–SDS assay was shown to correlate with the amount of truncated peptide observed from the reduced and alkylated peptide map of the engineered mAb.

Oligomerization of Transmembrane Alpha-Helices Modulated By C-terminal Boundary Residues

In studies of the structural biology of membrane proteins, the success of strategies based on the “divide-and-conquer” approach, where peptides are used to model the individual transmembrane (TM) α-helices of membrane proteins, depends upon the correct identification of the membrane-embedded TM α-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of the TM2 α-helix of myelin proteolipid protein (PLP). Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS) to study parent sequence KKKK-61AFQYVIYGTASFFFLYGALL-LAEG89-KKKK - along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and a hydrophobic mutant Leu-90 - we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is sufficient to change its intrinsic properties, with TM2 61-89 displaying only a monomeric form, but with principally dimers arising for the other three peptides. The findings suggest that deletion of critical C-terminal residue(s) tends to re-position the helix terminus toward the membrane-aqueous interface, and emphasize the potential influence of boundary residues on TM properties when utilizing peptides as models for TM α-helices. These finding may implicate a role for such residues in membrane protein folding and assembly.