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Protein Modifications (protein + modifications)
Selected AbstractsDetection of carbonyl-modified proteins in interfibrillar rat mitochondria using N, -aminooxymethylcarbonylhydrazino- D -biotin as an aldehyde/keto-reactive probe in combination with Western blot analysis and tandem mass spectrometryELECTROPHORESIS, Issue 6 2008Woon-Gye Chung Abstract There is now a large body of supporting data available that links oxidative modifications of proteins to a large number of diseases, degenerative disorders and aging. However, the detailed analysis of oxidative protein modifications remains challenging. Here, we report a new efficient method for identification of oxidatively modified proteins in complex biological samples which is based on the use of an aldehyde-reactive probe, N,-aminooxymethylcarbonylhydrazino- D -biotin (ARP), in combination with Western-type analyses and MS. The biotinylated hydroxylamine derivative forms a chemically stable oxime derivative with the aldehyde/keto group found in carbonyl-modified proteins. The biotin tag is detected by avidin affinity staining. ARP-positive proteins are subsequently subjected to in-gel trypsinization and MS/MS for protein identification. We demonstrate the usefulness of the method for the analysis of protein extracts obtained from interfibrillar heart mitochondria (IFM) from young and old rats. In this study, we identified as putative major protein targets of oxidative modifications the mitochondrial matrix protein, aconitase, the inner mitochondrial membrane protein, ADP/ATP translocase, and constituents of the electron transport chain complexes IV and V. An age-related increase of carbonyl levels was found for aconitase and ATP synthase. [source] Fast visible dye staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels compatible with matrix assisted laser desorption/ionization-mass spectrometryELECTROPHORESIS, Issue 7-8 2004Jung-Kap Choi Abstract A fast and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) compatible protein staining method in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1- and 2-D SDS-PAGE) is described. It is based on the counterion dye staining method that employs oppositely charged two dyes, zincon (ZC) and ethyl violet (EV) to form an ion-pair complex. The protocol, including fixing, staining and quick washing steps, can be completed in 1,1.5 h depending upon gel thickness. It has a sensitivity of 4,8 ng, comparable to that of colloidal Coomassie Brilliant Blue G (CBBG) staining with phosphoric acid in the staining solution. The counterion dye stain does not induce protein modifications that complicate interpretation of peptide mapping data from MS. Considering the speed, sensitivity and compatibility with MS, the counterion dye stain may be more practical than any other dye-based protein stains for routine proteomic researches. [source] Mass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp.FEBS JOURNAL, Issue 13 2009Nitrogenase Fe-protein modification was analyzed in the endophytic ,-proteobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore, a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide, indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach, we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosylations. [source] Lipid and protein changes in chilled sea salmon (Pseudopercis semifasciata): effect of previous rosemary extract (Rossmarinus officinalis L.) applicationINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 6 2009Valeria Tironi Summary The aim of this work was to analyse the effect of rosemary extract application (200 and 500 ppm) on lipid oxidation, colour and protein modifications during the chilled storage (1.0 ± 0.7 °C) of sea salmon (Pseudopercis semifasciata). Lipid oxidation and ,3-22:6 fatty acid content modification were prevented by the addition of rosemary extract. Analysis of interaction between lipid oxidation products and proteins by fluorescence showed no relationship between their temporal changes in the aqueous phase and the lipid oxidation evolution since a similar behaviour was observed in both absence and presence of antioxidant. Protein extractability, sodium dodecyl sulphate-polyacrylamide gel electrophoresis, differential scanning calorimetry and lysine content determinations revealed no differences between muscle untreated or treated with rosemary. Fluorescent compounds evolution in organic phase would be in relation with the appearance of lipid oxidation products. In addition, rosemary extract partially prevented the loss of red colour in chilled muscle. Although protein alterations could not be prevented, rosemary extract shows to be a promissory antioxidant in sea salmon muscle. [source] In silico modulation of apoptotic Bcl-2 proteins by mistletoe lectin-1: Functional consequences of protein modificationsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Tasneem A. Khwaja Abstract The mistletoe lectin-1 (ML-1) modulates tumor cell apoptosis by triggering signaling cascades through the complex interplay of phosphorylation and O -linked N -acetylglucosamine (O -GlcNAc) modification in pro- and anti-apoptotic proteins. In particular, ML-1 is predicted to induce dephosphorylation of Bcl-2-family proteins and their alternative O -GlcNAc modification at specific, conserved Ser/Thr residues. The sites for phosphorylation and glycosylation were predicted and analyzed using Netphos 2.0 and YinOYang 1.2. The involvement of modified Ser/Thr, and among them the potential Yin Yang sites that may undergo both types of posttranslational modification, is proposed to mediate apoptosis modulation by ML-1. J. Cell. Biochem. 103: 479,491, 2008. © 2007 Wiley-Liss, Inc. [source] Characterization of N -palmitoylated human growth hormone by in situ liquid,liquid extraction and MALDI tandem mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 6 2007Emmanuelle Sachon Abstract Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N, group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid,liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N -palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N -palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications. Copyright © 2007 John Wiley & Sons, Ltd. [source] Solid-state glycation of ,-lactoglobulin by lactose and galactose: localization of the modified amino acids using mass spectrometric techniquesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2004François Fenaille Abstract The Maillard reaction is commonly encountered during food processing or storage, and also in human nutrition, hence there is a need for analytical methodologies to identify and characterize the modified proteins. This paper reports specific methods using mass spectrometric techniques to localize protein modifications induced by lactose and galactose on ,-lactoglobulin (,-Lg) under solid-state glycation conditions. The extent of glycation was first determined by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The specific identification of lactose-modified amino acid residues was realized using both NanoESI-MS, NanoESI-MS/MS (neutral loss scanning modes) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) (with and without guanidination of lysine residues) on unfractionated digests. The results indicated that, after 8.25 h of incubation, the lysine residues were the main targets of lactose-induced modification. In addition to the 15 lysine residues, Leu1 (NH2 terminal) and the Arg124 were also found to be modified, thus leading to a total of 17 different modified amino acid residues (versus 15 found by LC/ESI-MS measurement). In a second set of experiments, different strategies consisting of constant neutral loss and precursor ion scanning were compared to characterize galactose-induced modifications. Owing to the high level of ,-Lg glycation, the combined use of these different strategies appeared to be necessary for determining the galactose-modified sites after 8.25 h of incubation. Thus, among the 22 galactose adducts deduced from the LC/ESI-MS measurement, apart from the N-terminal and classical lysine residues, we also observed a few arginine residues (Arg40, Arg124 and Arg148) that were modified, and also dialkylations on specific lysine residues (Lys47, Lys75). Copyright © 2003 John Wiley & Sons, Ltd. [source] Cerebral ischemia/stroke and small ubiquitin-like modifier (SUMO) conjugation , a new target for therapeutic intervention?JOURNAL OF NEUROCHEMISTRY, Issue 3 2008Wei Yang Abstract Transient cerebral ischemia/stroke activates various post-translational protein modifications such as phosphorylation and ubiquitin conjugation that are believed to play a major role in the pathological process triggered by an interruption of blood supply and culminating in cell death. A new system of post-translational protein modification has been identified, termed as small ubiquitin-like modifier (SUMO) conjugation. Like ubiquitin, SUMO is conjugated to the lysine residue of target proteins in a complex process. This review summarizes observations from recent experiments focusing on the effect of cerebral ischemia on SUMO conjugation. Transient global and focal cerebral ischemia both induced a rapid, dramatic and long-lasting rise in levels of SUMO2/3 conjugation. After transient focal cerebral ischemia, SUMO conjugation was particularly prominent in neurons located at the border of the ischemic territory where SUMO-conjugated proteins translocated to the nucleus. Many SUMO conjugation target proteins are transcription factors and sumoylation has been shown to have a major impact on the activity, stability, and cellular localization of target proteins. The rise in levels of SUMO-conjugated proteins is therefore likely to have a major effect on the fate of post-ischemic neurons. The sumoylation process could provide an exciting new target for therapeutic intervention. [source] Peroxynitrite scavenging activity of lithospermate B from Salvia miltiorrhizaJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2003Do You Soung ABSTRACT Peroxynitrite (ONOO,) is produced by the reaction of superoxide (O2,) with nitric oxide. ONOO, damages proteins through nitration or oxidation. For protection from ONOO, induced protein modifications, ONOO, scavengers should be supplemented. Evidence was obtained that lithospermate B extracted from Salvia miltiorrhiza showed the strongest scavenging activity among its constituents. Its ONOO, scavenging activity is via an electron donation mechanism. A dihydroxyl group and a double bond seem to be essential structure requirements. The data from the experiments further confirmed a protective effect of lithospermate B on bovine serum albumin and low-density lipoprotein against ONOO,. This study demonstrated that lithospermate B with hydroxyl groups and double bonds exerts an anti-nitration effect by scavenging ONOO,. [source] Lipid,protein modifications during ascorbate-Fe2+ peroxidation of photoreceptor membranes: protective effect of melatoninJOURNAL OF PINEAL RESEARCH, Issue 3 2006Margarita H. Guajardo Abstract:, The rod outer segment (ROSg) membranes are essentially lipoprotein complexes. Rhodopsin, the major integral protein of ROSg, is surrounded by phospholipids highly enriched in docosahexaenoic acid (22:6 n3). This fluid environment plays an important role for conformational changes after photo-activation. Thus, ROSg membranes are highly susceptible to oxidative damage. Melatonin synthesized in the pineal gland, retina and other tissues is a free radical scavenger. The principal aim of this work was to study the changes in the ROSg membranes isolated from bovine retina submitted to nonenzymatic lipid peroxidation (ascorbate-Fe2+ induced), during different time intervals (0,180 min). Oxidative stress was monitored by increase in the chemiluminescence and fatty acid alterations. In addition we studied the in vitro protective effect of 5 mm melatonin. The total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of melatonin. The docosahexaenoic acid content decreased considerably when the membranes were exposed to oxidative damage. This reduction was from 35.5 ± 2.9% in the native membranes to 12.65 ± 1.86% in those peroxidized during 180 min. In the presence of 5 mm melatonin we observed a content preservation of 22:6 n3 (23.85 ± 2.77%) at the same time of peroxidation. Simultaneously the alterations of membrane proteins under oxidative stress were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Loss of protein sulfhydryl groups and increased incorporation of carbonyl groups were utilized as biomarkers of protein oxidation. In membranes exposed to Fe2+ -ascorbate, we observed a decrease of protein thiols from 50.9 ± 3.38 in native membranes to 1.72 ± 2.81 nmol/mg of protein after 180 min of lipid peroxidation associated with increased incorporation of carbonyl groups into proteins from 7.20 ± 2.50 to 12.50 ± 1.12 nmol/mg of protein. In the SDS-PAGE we observed a decrease in the content of all the proteins, mainly rhodopsin, as a consequence of peroxidation. Melatonin, prevent both lipid peroxidation and protein oxidation. [source] Primary and secondary metabolism, and post-translational protein modifications, as portrayed by proteomic analysis of Streptomyces coelicolorMOLECULAR MICROBIOLOGY, Issue 4 2002A. R. Hesketh Summary The newly sequenced genome of Streptomyces coelicolor is estimated to encode 7825 theoretical proteins. We have mapped approximately 10% of the theoretical proteome experimentally using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Products from 770 different genes were identified, and the types of proteins represented are discussed in terms of their anno-tated functional classes. An average of 1.2 proteins per gene was observed, indicating extensive post-translational regulation. Examples of modification by N-acetylation, adenylylation and proteolytic processing were characterized using mass spectrometry. Proteins from both primary and certain secondary metabolic pathways are strongly represented on the map, and a number of these enzymes were identified at more than one two-dimensional gel location. Post-translational modification mechanisms may therefore play a significant role in the regulation of these pathways. Unexpectedly, one of the enzymes for synthesis of the actinorhodin polyketide antibiotic appears to be located outside the cytoplasmic compartment, within the cell wall matrix. Of 20 gene clusters encoding enzymes characteristic of secondary metabolism, eight are represented on the proteome map, including three that specify the production of novel metabolites. This information will be valuable in the characterization of the new metabolites. [source] Quantitative FRET Analysis With the E0GFP-mCherry Fluorescent Protein PairPHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2009Lorenzo Albertazzi Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein,protein interaction and even protein modifications in living cells. Here, we analyze the E0GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro. The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R0 = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E0GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo. Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo) reveals that this new pair can be used for very effective quantitative FRET imaging. [source] Island clustering analysis for the comparison of the membrane and the soluble protein fractions of human brain proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2008Kyung-Hoon Kwon Abstract A protein identified in multiple separate bands of a 1-D gel reflects variation in the molecular weight caused by alternative splicing, endoproteolytic cleavage, or PTMs, such as glycosylation or ubiquitination. To characterize such a protein distribution over the bands, we defined an entity called an ,island' as the band region including the bands of the same protein identified sequentially. We quantified the island distribution using a new variable called an Iscore. Previously, as described in Park et al.. (Proteomics 2006, 6, 4978,4986.), we analyzed human brain tissue using a multidimensional MS/MS separation method. Here, the new method of island analysis was applied to the previous proteome data. The soluble and membrane protein fractions of human brain tissue were reanalyzed using the island distribution. The proteome of the soluble fraction exhibited more variation in island positions than that of the membrane fraction. Through the island analysis, we identified protein modifications and protein complexes over the 1-D gel bands. [source] A general precursor ion-like scanning mode on quadrupole-TOF instruments compatible with chromatographic separationPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 1 2006Ricarda Niggeweg Abstract MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic samples is often hampered by the limited dynamic range of current analytical tools. Here we present a fast, sensitive, multiplexed precursor ion scanning mode , implemented on a quadrupole-TOF instrument , that allows the specific detection of any modified peptide or molecule that reveals itself by a specific fragment ion or pattern of fragment ions within a complex proteomic sample. The high mass accuracy of the TOF mass spectrometer is available for the marker ion specificity and the precursor ion mass determination. The method is compatible with chromatographic separation. Fragment ions and intact molecular ions are acquired quasi-simultaneously by continuously switching the collision energy between elevated and low levels. Using this technique many secondary modifications can be analyzed in parallel; however, the number of peptides carrying a specific modification that can be analyzed successfully is limited by the chromatographic resolution or, more generally, by the depth of the resolved time domain. [source] Differential adduction of proteins vs. deoxynucleosides by methyl methanesulfonate and 1-methyl-1-nitrosourea in vitro,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 4 2005Fagen Zhang The reactions of two model mutagenic and carcinogenic alkylating agents, N -methyl- N -nitrosourea (MNU) and methyl methanesulfonate (MMS), with proteins and deoxynucleosides in vitro, were investigated. The protein work used an approach involving trypsin digestion and high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS). This technique permitted identification of the specific location of protein adduction by both MNU and MMS with commercial apomyoglobin and human hemoglobin, under physiological conditions. MNU treatment resulted in predominantly carbamoylation adducts on the proteins, but in contrast only methylated protein adducts were found following treatment with MMS. Further analyses, using TurboSequest®, and the Scoring Algorithm for Spectral Analysis (SALSA), revealed that MNU carbamoylation was specific for modification of either the N-terminal valine or the free amino group in lysine residues of apomyglobin and human hemoglobin. However, MMS methylation modified the N-terminal valine and histidine residues of the proteins. Despite their clear differences in protein modifications, MNU and MMS formed qualitatively the same methylated deoxynucleoside adduct profiles with all four deoxynucleosides in vitro under physiological conditions. In light of their different biological potencies, where MMS is considered a ,super clastogen' while MNU is a ,super mutagen', these differences in reaction products with proteins vs. deoxynucleosides may indicate that these two model alkylating agents work via different mechanisms to produce their mutagenic and carcinogenic effects. Copyright © 2005 John Wiley & Sons, Ltd. [source] A hierarchical analysis of transcriptome alterations in intrauterine growth restriction (IUGR) reveals common pathophysiological pathways in mammals,THE JOURNAL OF PATHOLOGY, Issue 3 2007C Buffat Abstract Intra-uterine growth restriction (IUGR) is a frequent disease, affecting up to 10% of human pregnancies and responsible for increased perinatal morbidity and mortality. Moreover, low birth weight is an important cause of the metabolic syndrome in the adult. Protein depletion during the gestation of rat females has been widely used as a model for human IUGR. By transcriptome analysis of control and protein-deprived rat placentas, we were able to identify 2543 transcripts modified more than 2.5 fold (1347 induced and 1196 repressed). Automatic functional classification enabled us to identify clusters of induced genes affecting chromosome structure, transcription, intracellular transport, protein modifications and apoptosis. In particular, we suggest the existence of a complex balance regulating apoptosis. Among repressed genes, we noted several groups of genes involved in immunity, signalling and degradation of noxious chemicals. These observations suggest that IUGR placentas have a decreased resistance to external aggression. The promoters of the most induced and most repressed genes were contrasted for their composition in putative transcription factor binding sites. There was an over-representation of Znfinger (ZNF) proteins and Pdx1 (pancreatic and duodenal homeobox protein 1) putative binding sites. Consistently, Pdx1 and a high proportion of ZNF genes were induced at the transcriptional level. A similar analysis of ZNF promoters showed an increased presence of putative binding sites for the Tata box binding protein (Tbp). Consistently again, we showed that the Tbp and TBP-associated factors (Tafs) were up-regulated in IUGR placentas. Also, samples of human IUGR and control placentas showed that human orthologous ZNFs and PDX1 were transcriptionnally induced, especially in non-vascular IUGR. Immunohistochemistry revealed increased expression of PDX1 in IUGR human placentas. In conclusion, our approach permitted the proposition of hypotheses on a hierarchy of gene inductions/repressions leading to massive transcriptional alterations in the IUGR placenta, in humans and in rodents. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Proline Analogue of Nitrosourea as a New Cytotoxic ProdrugARCHIV DER PHARMAZIE, Issue 11 2009Anna Stankiewicz-Kranc Abstract Carmustine is frequently used as anticancer drug. High toxicity and low selectivity reduces the application of this drug. Though, there is a necessity to find new compounds characterized by similar therapeutic effects but a higher selectivity and safety. As a result, the proline analogue of nitrosourea, N -[N,-(2-bromophenyl)- N,-nitrosocarbamoyl]proline (AC), has been synthesized. The aim of this study was to compare the influence of carmustine and the proline analogue of nitrosourea on the antioxidant abilities of fibroblasts and leukemia cells, MOLT4. It was shown that carmustine as well as AC cause an increase in hydrogen peroxide concentration in normal and neoplastic cells. Incubation with both compounds led to a diminution of the activity of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, and reductase. Changes in activity / level of antioxidant parameters were accompanied by augmentation of lipid and oxidative protein modifications. In conclusion, carmustine and AC cause changes in the antioxidative system of normal and MOLT4 cells and are a reason of oxidative stress formation. [source] Investigating variables and mechanisms that influence protein integrity in low water content amorphous carbohydrate matricesBIOTECHNOLOGY PROGRESS, Issue 5 2009Jane F. Povey Abstract Biopharmaceutical proteins are often formulated and freeze dried in agents that protect them from deleterious reactions that can compromise activity and authenticity. Although such approaches are widely used, a detailed understanding of the molecular mechanisms of protein stabilization in low water content amorphous glasses is lacking. Further, whilst deterioration chemistries are well described in dilute solution, relatively little is known about the extent and mechanisms by which protein integrity is compromised in the glassy state. Here we have investigated the relationship between protein modification and rate thereof, with variation of pH, carbohydrate excipient, temperature and the glass transition temperature using a model protein, lysozyme. Mass spectrometry analysis and peptide mapping confirm that protein modifications do occur in the glassy state in a time-, temperature-, and carbohydrate excipient-dependent manner. There were clear trends between the buffer pH and the primary modification detected (glycation). Most importantly, there were differences in the apparent reactivities of the lysine residues in the glass compared with those previously determined in solution, and therefore, the well-characterized solution reactivity of this reaction cannot be used to predict likely sites of modification in the glassy state. These findings have implications for (i) the selection and combinations of formulation components, particularly with regard to glycation in the glassy state, and (ii) the design of procedures and methodologies for the improvement of protein stability in the glassy state. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||||||||