Assessment method for deamidation in proteins using carboxylic acid derivatization-liquid chromatography-tandem mass spectrometry.

An analytical method for the degree of protein deamidation has been developed by using carboxy group derivatization and liquid chromatography-tandem mass spectrometry (LCMS/MS). The fragment peptides (LGEYGFQNALIVR and YNGVFQECCQAEDK) obtained by digesting bovine serum albumin (BSA) with trypsin and their asparagine deamidated peptides (LGEYGFQDALIVR and YDGVFQECCQAEDK) were selected as model peptides, and their carboxy groups were derivatized with ethylamine. This derivatization enabled a clear distinction between natural peptides and deamidated peptides by mass, allowing for facile distinction by LCMS/MS before and after deamidation. Good linearity was confirmed for four peptides used in this study via isotope dilution mass spectrometry, showing that protein deamidation can be evaluated by the present method. To confirm the validity of this method for the evaluation of deamidation, natural peptides and deamidated peptides were mixed in arbitrary ratios, and degree of deamidation in these solution was analyzed. This confirmed that accurate evaluation was possible at deamidation degree values of ca. 10 %, 5 %, 2.5 %, and 1 %. Additionally, an accelerated storage test of BSA demonstrated that the deamidation of asparagine at position 404 of BSA progressed by 4 % in 9 weeks at 40 °C and pH 8 in the dark, and that the deamidation process can be traced over time.

A Review of Potential Marine-derived Hypotensive and Anti-obesity Peptides.

Bioactive peptides are food derived components, usually consisting of 3-20 amino acids, which are inactive when incorporated within their parent protein. Once liberated by enzymatic or chemical hydrolysis, during food processing and gastrointestinal transit, they can potentially provide an array of health benefits to the human body. Owing to an unprecedented increase in the worldwide incidence of obesity and hypertension, medical researchers are focusing on the hypotensive and anti-obesity properties of nutritionally derived bioactive peptides. The role of the renin-angiotensin system has long been established in the aetiology of metabolic diseases and hypertension. Targeting the renin-angiotensin system by inhibiting the activity of angiotensin-converting enzyme (ACE) and preventing the formation of angiotensin II can be a potential therapeutic approach to the treatment of hypertension and obesity. Fish-derived proteins and peptides can potentially be excellent sources of bioactive components, mainly as a source of ACE inhibitors. However, increased use of marine sources, poses an unsustainable burden on particular fish stocks, so, the underutilized fish species and by-products can be exploited for this purpose. This paper provides an overview of the techniques involved in the production, isolation, purification, and characterization of bioactive peptides from marine sources, as well as the evaluation of the ACE inhibitory (ACE-I) activity and bioavailability.

Two novel antioxidant nonapeptides from protein hydrolysate of skate (Raja porosa) muscle.

In the current study, the preparation conditions of neutrase hydrolysate (SMH) from skate (Raja porosa) muscle protein were optimized using orthogonal L9(3)4 tests, and R values indicated that pH was the most important factor affecting HO· scavenging activity of SMH. Under the optimum conditions of pH 7.0, enzymolysis temperature 60 °C, enzyme/substrate ratio (E/S) 2%, and enzymolysis time 5 h, EC50 of SMH on HO· was 2.14 ± 0.17 mg/mL. Using ultrafiltration, gel filtration chromatography, and RP-HPLC, two novel antioxidant nonapeptides (SP-A and SP-B) were isolated from SMH and their amino acid sequences were found to be APPTAYAQS (SP-A) and NWDMEKIWD (SP-B) with calculated molecular masses of 904.98 Da and 1236.38 Da, respectively. Both showed strong antioxidant activities. SP-A and SP-B exhibited good scavenging activities on HO· (EC50 0.390 and 0.176 mg/mL), DPPH· (EC50 0.614 and 0.289 mg/mL), and O2-· (EC50 0.215 and 0.132 mg/mL) in a dose-dependent manner. SP-B was also effective against lipid peroxidation in the model system. The aromatic (2Trp), acidic (2Asp and Glu), and basic (Lys) amino acid residues within the sequences of SP-B might account for its pronounced antioxidant activity. The results of this study suggested that protein hydrolysate and peptides from skate muscle might be effective as food additives for retarding lipid peroxidation occurring in foodstuffs.

Purification and identification of angiotensin I-converting enzyme-inhibitory peptides from apalbumin 2 during simulated gastrointestinal digestion.

BACKGROUND: Bee larvae are considered to be an important reservoir for proteins. However, little attention has been paid to the release of potential bioactive peptides from bee larva proteins. In this study the major protein in bee larvae was hydrolyzed in vitro by gastrointestinal enzymes. The peptide profile of the hydrolysis was characterized by gel filtration chromatography and tricine-SDS-PAGE. Furthermore, the bioactive peptide was isolated and identified by Q-TOF-MS/MS. RESULTS: The major bee larva protein was identified as apalbumin 2 and was more digestible into peptides with molecular weights lower than 3 kDa. The hydrolysate obtained after 3 h of digestion exhibited angiotensin I-converting enzyme (ACE)-inhibitory activity and was purified sequentially by gel filtration and RP-HPLC. The molecular weights of peptide fractions with ACE-inhibitory activity were distributed between 0.5 and 1.5 kDa. A novel peptide with highest ACE-inhibitory activity (IC50 54.9 µmol L(-1) ) was purified by further RP-HPLC. The amino acid sequence of this peptide was identified as LLKPY (632.40 Da). CONCLUSION: ACE-inhibitory peptides could be formed from bee larvae through gastrointestinal digestion. The most active peptide (LLKPY) is potentially useful as a therapeutic agent in treating hypertension.

Identification of bitter peptides in aged cheddar cheese.

The compounds responsible for the bitter taste of aged "sharp" Cheddar cheese were characterized. Sensory-guided fractionation techniques using gel permeation chromatography and multi-dimension semi-preparative reversed-phase high-performance liquid chromatography revealed the presence of multiple bitter compounds. The compounds with the highest perceived bitterness intensity were identified by tandem mass spectrometry de novo peptide sequencing as GPVRGPFPIIV, YQEPVLGPVRGPFPI, MPFPKYPVEP, MAPKHKEMPFPKYPVEPF, and APHGKEMPFPKYPVEPF; all originated from ß-casein. Subsequent quantitative liquid chromatography-tandem mass spectrometry analysis reported that the concentrations of GPVRGPFPIIV, YQEPVLGPVRGPFPI, and MPFPKYPVEP increased during maturation by 28.7-, 3.1-, and 1.8-fold, respectively. When directly compared to young "mild" Cheddar, APHGKEMPFPKYPVEPF was reported only in the sharp Cheddar cheese, whereas the concentration of MAPKHKEMPFPKYPVEPF did not change. Further taste re-engineering sensory experiments confirmed the importance of the identified peptides to the bitterness of sharp Cheddar. The bitter intensity of the aged "sharp" Cheddar model (mild Cheddar with equivalent concentrations of the five bitter peptides in the sharp sample) was rated as not significantly different from the authentic sharp Cheddar cheese. Among the five peptides, GPVRGPFPIIV was reported to be the main contributor to the bitterness intensity of sharp Cheddar. Furthermore, a difference from control sensory test also confirmed the significance of the bitter taste to the overall perception of aged Cheddar flavor. The sharp Cheddar model was reported to be significantly more similar to aged "sharp" Cheddar in comparison to the young "mild" Cheddar cheese sample.

Accuracy of ELISA detection methods for gluten and reference materials: a realistic assessment.

The determination of prolamins by ELISA and subsequent conversion of the resulting concentration to gluten content in food appears to be a comparatively simple and straightforward process with which many laboratories have years-long experience. At the end of the process, a value of gluten, expressed in mg/kg or ppm, is obtained. This value often is the basis for the decision if a product can be labeled gluten-free or not. On the basis of currently available scientific information, the accuracy of the obtained values with commonly used commercial ELISA kits has to be questioned. Although recently several multilaboratory studies have been conducted in an attempt to emphasize and ensure the accuracy of the results, data suggest that it was the precision of these assays, not the accuracy, that was confirmed because some of the underlying assumptions for calculating the gluten content lack scientific data support as well as appropriate reference materials for comparison. This paper discusses the issues of gluten determination and quantification with respect to antibody specificity, extraction procedures, reference materials, and their commutability.

Hypoallergenicity of various miso pastes manufactured in Japan.

Miso paste (miso), a fermented soybean food, is popular in Japan and other Asian countries. However, the soybean is known to induce an allergenic response in some individuals. In the present study, we evaluated the allergenicity of various kinds of miso available in Japan. Total proteins were extracted from Amakuti-kome miso, Karakuti-kome miso, Mugi-miso and Mame-miso, and the protein profiles were analyzed. The major protein bands detected in the intact soybean extract were not present in any of the miso samples, which instead showed various low molecular weight protein bands of approximately 10-25 kDa. The existence levels of six major soybean allergens were determined by Western blotting using specific antibodies. We found that the allergen levels varied among miso and allergen types; however, allergen levels were consistently lower in miso than in the soybean extract. We obtained similar results for IgE-ELISA experiments using serum IgE from soybean allergy patients. Taken together, these results indicate that compared to soybean extract, various types of miso contain small quantities of intact soybean allergens. Additionally, several lines of evidence indicated that the allergen levels were exceptionally low in the dark-colored Karakuti-kome miso and Mame-miso, which are produced with relatively long fermentation periods, suggesting that the duration of fermentation might be a key factor in the hypoallergenicity of miso.

Isolation and characterization of pepsin-solubilized collagen from the integument of sea cucumber (Stichopus vastus).

BACKGROUND: Sea cucumber (Stichopus vastus) is considered an underutilized resource, since only its stomach and intestines are eaten raw as salad in a few countries and the remaining parts, especially the integument rich in collagen, is discarded. Hence a valuable by-product having potential nutraceutical and pharmaceutical applications is wasted. In the present investigation, pepsin-solubilized collagen (PSC) from the integument of S. vastus was isolated, purified and characterized. RESULTS: Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the purified collagen was of type I, consisting of three α1 chains of approximately 122 kDa each. The peptide map of PSC digested by V8 protease was different from that of calf skin type I collagen. Fourier transform infrared spectroscopy revealed that the triple helical structure was well preserved in isolated collagen. The denaturation temperature of PSC was 21.23 °C and showed good gel-forming capability at pH 6.5 and 300 mmol L⁻¹ NaCl. CONCLUSION: It is inferred that the collagen isolated from S. vastus integument has potential for use as an alternative to land-based mammalian collagen in food, nutraceuticals and pharmaceutical industries.

Improving surface functional properties of tofu whey-derived peptides by chemical modification with fatty acids.

Effect of acylation with saturated fatty acids on surface functional properties of tofu whey-derived peptides was investigated. Tofu whey (TW) and soy proteins (7S, 11S, and acid-precipitated soy protein [APP]) were hydrolyzed by Protease M 'Amano' G, and resulting peptide mixtures were acylated with esterified fatty acids of different chain length (6C to 18C) to form a covalent linkage between the carboxyl group of fatty acid and the free amino groups of peptide. Acylation significantly (P < 0.05) increased emulsifying properties of 7S, 11S, and APP peptides independent of fatty acid chain length. Acylation decreased water binding capacity although oil binding capacity of acylated tofu whey ultra filtered fraction (UFTW < 3 kDa), 7S- and 11S-peptides were improved compared to native peptides. 7S peptides acylated with long chain fatty acids had shown significant higher surface hydrophobicity as in contrast with acylated UFTW < 3 kDa and APP peptides. Fluorescence spectra studies revealed structural conformation of acylated soy peptides as compared to native peptides. This study shows that chemical modification with fatty acids can further affect functional properties of soy proteins.

Review: Production and functionality of active peptides from milk.

In recent years, research on the production of active peptides obtained from milk and their potential functionality has grown, to a great extent. Bioactive peptides have been defined as specific protein fragments that have a positive impact on body functions or conditions, and they may ultimately have an influence on health. Individual proteins of casein or milk-derived products such as cheese and yogurt have been used as a protein source to study the isolation and activity of peptides with several applications. Currently, the milk whey waste obtained in the production of cheese also represents a protein source from which active peptides could be isolated with potential industrial applications. The active properties of milk peptides and the results found with regard to their physiological effects have led to the classification of peptides as belonging to the group of ingredients of protein nature, appropriate for use in functional foods or pharmaceutical formulations. In this study, the main peptides obtained from milk protein and the past research studies about its production and biological activities will be explained. Second, an analysis will be made on the methods to determinate the biological activities, the separation of bioactive peptides and its structure identification. All of these form the base required to obtain synthetic peptides. Finally, we explain the experimental animal and human trials done in the past years. Nevertheless, more research is required on the design and implementation of equipment for the industrial production and separation of peptides. In addition, different authors suggest that more emphasis should therefore be given to preclinical studies, proving that results are consistent and that effects are demonstrated repeatedly by several research human groups.

In vitro gastrointestinal digestion of the major peach allergen Pru p 3, a lipid transfer protein: molecular characterization of the products and assessment of their IgE binding abilities.

A simulated gastrointestinal digestion has been carried out on purified peach lipid transfer protein, one of the main allergens among the population of the Mediterranean area and the major allergen of peach allergic patients. The percentage of intact protein, after extensive digestion, measured by comparison with a non-digestible peptide analogue used as internal standard, was found to be about one-third of the original protein content. The peptides formed in digested fraction were characterized by means of LC/MS. The products of the digestion essentially derived from trypsin action, whereas the protein appeared to be resistant to pepsin and chymotrypsin. The identified peptides could be classified as low molecular weight and high molecular weight peptides. The latter consisted of the full protein, with the disulfide bridges still intact, deprived of the smaller peptides. The different digestion products, including the high and low molecular weight peptides, were purified by LC and assessed, together with the intact protein, by dot-blot analysis with sera of allergic patients, allowing to estimate their potential allergenicity. The intact protein and the high molecular weight peptides were found to be recognized by patients' sera, whereas the small peptides were found to be not reactive.

Assessment of antibody fragmentation by reversed-phase liquid chromatography and mass spectrometry.

Antibody fragmentation in the hinge region and other regions, and the impact of pH on the level and pattern of antibody fragmentation were investigated by reversed-phase (RP) liquid chromatography and mass spectrometry (LC-MS). Extensive fragmentation was observed in the hinge and in regions other than the hinge of a recombinant monoclonal antibody that was incubated in buffers of various pH at 40 degrees C for 10 weeks. Peptide bonds that were susceptible to hydrolysis were located mainly around the domain-domain interfaces close to or in the loop structures. The sites as well as the level of peptide bond hydrolysis were affected by the buffer pH. In agreement with previous findings when only the hinge region fragmentation was monitored, pH 6 was optimal for slowing down antibody fragmentation in regions other than the hinge. It also demonstrated that analysis by RPLC-MS provided a better assessment of the susceptible regions of recombinant monoclonal antibodies than size-exclusion chromatography (SEC) followed by fraction collection and mass spectrometry identification.

Development of homogeneous 384-well high-throughput screening assays for Abeta1-40 and Abeta1-42 using AlphaScreen technology.

Amyloid beta (Abeta) peptides are the major constituent of amyloid plaques, one of the hallmark pathologies of Alzheimer's disease. Accurate and precise quantitation of these peptides in biological fluids is a critical component of Alzheimer's disease research. The current most established assay for analysis of Abeta peptides in preclinical research is enzyme-linked immunosorbent assay (ELISA), which, although sensitive and of proven utility, is a multistep, labor-intensive assay that is difficult to automate completely. To overcome these limitations, the authors have developed and optimized simple, sensitive, homogeneous 384-well assays for Abeta1-42 and Abeta1-40 using AlphaScreen technology. The assays are capable of detecting Abeta peptides at concentrations <2 pg/mL and, using a final assay volume of 20 microL, routinely generate Z' values >0.85. The AlphaScreen format has the following key advantages: substantially less hands-on time to run, easier to automate, higher throughput, and less expensive to run than the traditional ELISA. The results presented here show that AlphaScreen technology permits robust, efficient, and cost-effective quantitation of Abeta peptides.

An assessment of different filter systems for extracorporeal elimination of bivalirudin: an in vitro study.

IMPLICATIONS: Bivalirudin is a new, direct thrombin inhibitor. We investigated the extracorporeal elimination rate of different hemofilters and one plasmapheresis filter for bivalirudin. Our data show that bivalirudin can be effectively eliminated via hemofiltration and plasmapheresis, although there were significant differences in the elimination rates among the filter systems investigated.

The skeletal muscle ryanodine receptor identified as a molecular target of [3H]azidodantrolene by photoaffinity labeling.

Dantrolene is a skeletal muscle relaxant which acts by inhibiting intracellular Ca(2+) release from sarcoplasmic reticulum (SR). It is used primarily in the treatment of malignant hyperthermia (MH), a pharmacogenetic sensitivity to volatile anesthetics resulting in massive intracellular Ca(2+) release. Determination of the site and mechanism of action of dantrolene should contribute to the understanding of the regulation of intracellular Ca(2+) release in skeletal muscle. Photoaffinity labeling of porcine SR with [(3)H]azidodantrolene, a photoactivatable analogue of dantrolene, has identified a 160 kDa SR protein with immunologic cross-reactivity to skeletal muscle ryanodine receptor (RyR) as a possible target [Palnitkar et al. (1999) J. Med. Chem. 42, 1872-1880]. Here we demonstrate specific, AMP-PCP-enhanced, [(3)H]azidodantrolene photolabeling of both the RyR monomer and a 160 or 172 kDa protein in porcine and rabbit SR, respectively. The 160/172 kDa protein is shown to be the NH(2)-terminus of the RyR cleaved from the monomer by an endogenous protease activity consistent with that of n-calpain. MALDI-mass spectrometric analysis of the porcine 160 kDa protein identifies it as the 1400 amino acid NH(2)-terminal fragment of the skeletal muscle RyR reportedly generated by n-calpain [Shevchenko et al. (1998) J. Membr. Biol. 161, 33-34]. Immunoprecipitation of solubilized, [(3)H]azidodantrolene-photolabeled SR protein reveals that the cleaved 160/172 kDa protein remains associated with the C-terminal, 410 kDa portion of the RyR. [(3)H]Dantrolene binding to both the intact and the n-calpain-cleaved channel RyR is similarly enhanced by AMP-PCP. n-Calpain cleavage of the RyR does not affect [(3)H]dantrolene binding in the presence of AMP-PCP, but depresses drug binding in the absence of nucleotide. These results demonstrate that the NH(2)-terminus of the RyR is a molecular target for dantrolene, and suggest a regulatory role for both n-calpain activity and ATP in the interaction of dantrolene with the RyR in vivo.

[Is the assessment of prothrombin fragment F1+2 for monitoring of heparin therapy in patients with deep vein thrombosis useful?]. / Ist die Bestimmung von Prothrombinfragmenten F1+2 zur Kontrolle der Heparintherapie bei Patienten mit tiefer Venenthrombose nützlich?

Prothrombin fragments (F1+2) can be used to monitor oral anticoagulation; their generation is also suppressed by heparin. We studied the course of F1+2, aPTT, heparin doses administered and heparin concentrations, as well as prothrombin time, during the first 7 days of heparin therapy (target aPTT 70 s) and overlapping oral anticoagulation (target INR 2-3) in 26 patients routinely treated for deep venous thrombosis (deep venous thrombosis +/- pulmonary embolism, 23 patients) or pulmonary embolism with a history of recurring deep venous thrombosis (3 patients). Although we found significant suppression of F1+2 between all pre- and post-treatment values, a transient, significant increase was seen on days 2-4. The amount of heparin given was stable from day 1-5; the fact of a transient increase in F1+2 might thus indicate that heparin doses routinely used are too low for the treatment of hypercoagulability in deep venous thrombosis in patients. We also found a decrease in heparin concentration on day 2, which may be explicable by changed pharmacokinetics of heparin in individuals with deep venous thrombosis. In conclusion, F1+2 may be useful for monitoring heparin treatment and oral anticoagulation in deep venous thrombosis patients from a laboratory point of view. However, larger studies are necessary to confirm these results. It remains to be clarified whether monitoring of anticoagulation by molecular markers might improve therapy of deep venous thrombosis. Such studies are in progress.

Quantitative analysis of cyanogen bromide-cleaved peptides for the assessment of type I: type II collagen ratios in equine articular repair tissue.

Cyanogen bromide was used to solubilise and specifically fragment purified equine Type I and II collagen and equine articular surface repair tissue. The resultant peptides were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and quantified by densitometric scanning. Measurement of the relative amounts of the peptides alpha 2(I) CB3, 5 and alpha 1(II)CB10 provided an accurate method of establishing the ratio of Type I to Type II collagen in mixtures of purified equine collagens. The method was sensitive to 6% Type II collagen when the band areas were corrected for peptide molecular weight and the number of chains in the parent tropocollagen molecule which contain that particular peptide. Use of this technique showed that repair tissue in full thickness osteochondral defects in the dorso-distal margins of the intermediate carpal bones of ponies did not contain detectable amounts of Type II collagen 11 weeks after defect induction.

Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry.

Bovine rhodopsin has been reported to be S-palmitylated at cysteines 322 and 323 (Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A.S. (1988) FEBS Lett. 230, 1-5). Using a combination of enzymatic and chemical cleavage techniques in conjunction with tandem mass spectrometry, the sites of incorporation of the palmityl groups are shown. Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327). A bis-S-palmitylated synthetic standard had the same retention time by reversed-phase high performance liquid chromatography as the isolated peptide and the same molecular weight (MH+1511.7) by liquid secondary ion mass spectrometry. Dithiothreitol reduction of both the isolated and the synthetic peptide cleaved the two thioester-linked palmityl groups to produce reduction products of the same appropriately decreased molecular weight (MH+1035.5). Tandem mass spectrometry of the isolated and the synthetic peptide identified the sites of attachment of the palmityl groups on cysteines 322 and 323. These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors.

A biological price of antibiotic resistance: major changes in the peptidoglycan structure of penicillin-resistant pneumococci.

Pneumococcal strains with greatly elevated levels of resistance to penicillin have by now been described with increasing frequency worldwide. The mechanism of antibiotic resistance in these strains involves the molecular remodeling of cell wall synthetic enzymes (penicillin binding proteins). We have now analyzed the peptidoglycan structures of 10 penicillin-susceptible and 10 penicillin-resistant clinical isolates (4 of intermediate and 6 of high level resistance) with a high-resolution HPLC technique. Cell wall peptidoglycan of the susceptible strains contained monomeric and oligomeric forms of primarily (70% or more) linear stem peptides with the sequence of L-Ala-D-iGln-L-Lys-D-Ala (where iGln is isoglutamine). In contrast, the major peptide species (70% or more) of resistant cell walls were abnormal branched-stem peptides carrying Ala-Ser or Ala-Ala dipeptides on the epsilon-amino groups of the stem peptide lysine residues. The structural alteration in the peptidoglycan was not related to serotype, date, or site of isolation but showed strong correlation with penicillin resistance and was cotransformed with high-level penicillin resistance during genetic transformation. We suggest that the remodeling of the active site of penicillin binding proteins in the resistant bacteria, which results in the reduced affinity for penicillin, also changes the substrate preference of these enzymes for the more hydrophobic branched peptides (instead of linear peptides) for cell wall synthesis.

Assessment of the number of free cysteines and isolation and identification of cystine-containing peptides from acetylcholine receptor.

The number of free cysteines in each polypeptide of acetylcholine receptor from the electric organ of Torpedo californica has been assessed by alkylating the native protein with N-ethylmaleimide and iodoacetamide during homogenization of the tissue and alkylating the polypeptides with N-ethylmaleimide as they were unfolded in solutions of dodecyl sulfate. The cysteines unavailable for alkylation could be accounted for as specific cystines, connecting positions in the amino acid sequences of the individual polypeptides. Unreduced, alkylated polypeptides of acetylcholine receptor were digested with thermolysin or trypsin. Cystine-containing peptides in the chromatograms of the digests were identified electrochemically by the use of a dual gold/mercury electrode. Three thermolytic peptides and three tryptic peptides have been isolated from these digests and shown to contain intact cystines that were originally present in the native protein. The majority of these peptides contained an intact, intramolecular cystine connecting two cysteines in locations homologous to cysteines 128 and 142 from the alpha polypeptide. Each of these cystines from each of the polypeptides of acetylcholine receptor was isolated in at least one peptide, respectively. Each of these cystine-containing peptides also contained glucosamine. It can be concluded that each asparagine in the sequence Asn-Cys-Thr/Ser, which occurs in the respective, homologous location in every polypeptide, is glycosylated even though a cystine sits between the asparagine and the threonine or serine. In addition, the existence of the cystine connecting the adjacent cysteines, alpha 192 and alpha 193, in the alpha subunit of acetylcholine receptor [Kao, P. N., & Karlin, A. (1986) J. Biol. Chem. 261, 8085-8088] has been confirmed.