Evaluation of Glycated Globins by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Lapolla, Annunziata; Fedele, Domenico; Plebani, Mario; Aronica, Rosaria; Garbeglio, Massimo; Seraglia, Roberta; D’Alpaos, Martina; Traldi, Pietro

doi:10.1093/clinchem/45.2.288

Cited by 37 publications

(13 citation statements)

References 14 publications

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“…The higher‐molecular‐mass contaminant species (324 Da greater than the native mass) can be structurally assigned to protein modification resulting from the condensation of two units of glucose (or fructose) with a primary amino group on the surface of the protein. This non‐enzymic reaction has been well characterized and the mass change observed here (162 Da per glucose unit) is consistent with those previously reported for the glycation of proteins in the presence of relatively high concentrations (0.25 M) of glucose and fructose [17–19]. This di‐glycated contaminant adduct was always observed in both native and unheated control samples of β‐LGa and could not therefore be due to the formulation process.…”

Section: Resultssupporting

confidence: 90%

Mechanisms of protein modification during model anti‐viral heat‐treatment bioprocessing of β‐lactoglobulin variant A in the presence of sucrose

Smales

Pepper

James

2000

Biotech and App Biochem

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To ensure the safety of plasma and recombinant therapeutic proteins, heat treatment is routinely applied to these biopharmaceuticals as a means of virus inactivation. However, to maintain protein integrity during heat treatment it is necessary to use high concentrations of thermostabilizing excipients, such as sucrose, in order to prevent protein damage. In this study we describe the covalent modifications inferred to a model protein, beta-lactoglobulin A, that occur during typical and extended anti-viral heat treatments. The chemical derivation and mechanisms by which these modifications arise are addressed. Heat treatment initiated hydrolysis of sucrose to glucose and fructose, which in turn were degraded to glyoxal. Glyoxal and the free reducing sugars reacted with free amino groups in beta-lactoglobulin A to yield Maillard glycation adducts and advanced glycation end products (AGEs). The major mechanism for AGE formation was via degradation of glucose-derived Schiff-base adducts. Heat treatment and glycation of beta-lactoglobulin A resulted in thiol-disulphide interchange reactions leading to protein oligomerization. A small population of beta-lactoglobulin A non-disulphide-linked dimers were also observed with increasingly harsh heat treatments. These findings have implications for (i) improvements in the safety and efficacy of heat-treated protein biopharmaceuticals and (ii) our understanding of the mechanisms of protein glycation and AGE adduct formation.

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Section: Resultssupporting

confidence: 90%

Mechanisms of protein modification during model anti‐viral heat‐treatment bioprocessing of β‐lactoglobulin variant A in the presence of sucrose

Smales

Pepper

James

2000

Biotech and App Biochem

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“…1 ) and 15,866 were the doubly (2H+) and singly (1H+) charged forms of hemoglobin β subunit (Table 2 ). Interestingly, glycated forms of hemoglobin β subunit, previously characterized with MALDI-TOF by Lapolla et al [ 20 , 21 ], could also be detected on spectra of atrophic patients. Protonated molecules produced by the condensation of one glucose molecule on the hemoglobin β subunit could be observed at m/z 8033 (Fig.…”

Section: Resultsmentioning

confidence: 97%

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

et al. 2016

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BackgroundNonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing.MethodsThirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF–MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann–Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC–MS/MS, nephelometry, western blotting or ELISA assays.ResultsFrom the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF–MS using CM10 and IMAC-Cu2+ ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin β subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls.ConclusionTwo proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1019-1) contains supplementary material, which is available to authorized users.

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“…3 By means of this approach, higher glycation levels of human serum albumin (HSA) and immunoglobulin G were observed for pateints with poorly controlled diabetes than those determined for healthy subjects and well-controlled diabetics. 4,5 High levels of AGE-modified proteins activate, in vivo, a macrophage response with consequent internalization and digestion: These processes generate a series of AGE-modified (and, consequently, highly reactive) peptides, exhibiting the same toxic activity of AGEs, and these peptides are believed responsible for the tissue modification observed in chronic complications of diabetes (for their higher production) and end-stage renal disease (for their accumulation). 6 The direct detection of these species in serum is a difficult task, due to the complexity of serum and to the AGE/peptide levels, which are orders of magnitude lower than those of circulating proteins as, for example, HSA, immunoglobulins, and transferrin.…”

Section: Introductionmentioning

confidence: 99%

“…For this goal, matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS)2 is highly effective, allowing evaluation of the number of glucose molecules condensed on a specific protein 3. By means of this approach, higher glycation levels of human serum albumin (HSA) and immunoglobulin G were observed for pateints with poorly controlled diabetes than those determined for healthy subjects and well‐controlled diabetics 4,5…”

Section: Introductionmentioning

confidence: 99%

Advanced Glycation End Products/Peptides: An in Vivo Investigation

Lapolla

Fedele

Reitano

et al. 2005

Annals of the New York Academy of Sciences

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Advanced glycation end products/peptides (AGE/peptides) originate by in vivo enzymatic digestion of nonenzymatically glycated proteins, which are produced by reaction of glucose with primary amino groups present in the protein chain following the Maillard pattern. AGE/peptides are highly reactive species and can interact with tissue and circulating proteins, leading to tissue modification and impaired protein functionality. Serum levels of AGE/peptides are reported to be particularly high in diabetes (in terms of higher production) or in end-stage renal disease (in terms of accumulation). For these reasons, their structural identification is of high interest, giving information on their relationship with the pathological state and allowing the design of possible therapeutic interventions. We report here some preliminary results obtained by liquid chromatography/electrospray ionization/mass spectrometry (LC/ESI/MS) and matrix-assisted laser desorption ionization MS (MALDI-MS) investigations carried out on the low-molecular-weight serum peptide fraction from 10 healthy subjects, 10 patients with poorly controlled diabetes, and 10 patients with end-stage nephropathy.

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Evaluation of Glycated Globins by Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Cited by 37 publications

References 14 publications

Mechanisms of protein modification during model anti‐viral heat‐treatment bioprocessing of β‐lactoglobulin variant A in the presence of sucrose

Mechanisms of protein modification during model anti‐viral heat‐treatment bioprocessing of β‐lactoglobulin variant A in the presence of sucrose

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

Advanced Glycation End Products/Peptides: An in Vivo Investigation

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