Identification of Modified Proteins by Mass Spectrometry

Sickmann, Albert; Mreyen, Marcus; Meyer, Helmut E.

doi:10.1080/15216540214314

Cited by 36 publications

(20 citation statements)

References 20 publications

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“…Different degrees of glycosylation at a single site in a single protein can result in ''trains'' of protein spots that separate on the basis of different isoelectric point and/or molecular mass in 2DE gels (Sickmann et al, 2002). Moreover, location of the detected peptides in the alignment of MAP1 and MAP1-1 proteins showed predicted glycosylation sites in the different sequences which might be responsible for the differences in migration detected in 2DE gels.…”

Section: Discussionmentioning

confidence: 99%

Host cell-specific protein expression in vitro in Ehrlichia ruminantium

Postigo

Taoufik

Bell‐Sakyi

et al. 2008

Veterinary Microbiology

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Ehrlichia ruminantium, a tick-transmitted pathogen, is the causative agent of heartwater in ruminants. In this study, a proteomic approach was used to identify host cell-specific E. ruminantium proteins encoded by the map1 multigene family, expressed in vitro in bovine endothelial and tick cell cultures. Two-dimensional gel electrophoresis combined with mass spectrometry analysis was used to establish the identities of immunodominant proteins. Proteins extracted from E. ruminantiuminfected endothelial cells were shown to be products of the map1 gene, whereas tick cell-derived E. ruminantium proteins were products of a different gene, map1-1. The expressed proteins were found to be glycosylated. Differential expression of MAP1 family proteins in vitro in mammalian and tick cell cultures indicates that the map1 multigene family might be involved in the adaptation of E. ruminantium to the mammalian host and vector tick. #

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Section: Discussionmentioning

confidence: 99%

Host cell-specific protein expression in vitro in Ehrlichia ruminantium

Postigo

Taoufik

Bell‐Sakyi

et al. 2008

Veterinary Microbiology

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“…Bands of interest were cut out of the SDS gel, digested with trypsin, and further processed and analyzed by matrixassisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry using a Reflex-III equipped with a SCOUT 384 ion source (Bruker Daltonik, Bremen, Germany) (13,14). The identification of the proteins on the basis of the MALDI-mass fingerprint spectra combined with a database search was performed as previously described in detail (13,14).…”

Section: Methodsmentioning

confidence: 99%

“…The identification of the proteins on the basis of the MALDI-mass fingerprint spectra combined with a database search was performed as previously described in detail (13,14).…”

Section: Methodsmentioning

confidence: 99%

Antileukoproteinase: Modulation of neutrophil function and therapeutic effects on anti–type II collagen antibody–induced arthritis

Sehnert¹,

Cavcic²,

Böhm³

et al. 2004

Arthritis & Rheumatism

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Objective. Antileukoproteinase (ALP) is a physiologic inhibitor of granulocytic serine proteases. The present study was undertaken to investigate its therapeutic benefit in an antibody-transfer model of erosive polyarthritis and to elucidate its potential to interfere with immune complex-dependent inflammatory pathways.Methods. Arthritis development was induced in male (BALB/c ؋ B10.Q)F 1 mice by intravenous injection of two monoclonal antibodies specific for type II collagen and was quantified by clinical scoring and histopathology. Arthritis severity was assessed in a cohort of mice under systemic treatment with recombinant human ALP (daily doses of 0.1 mg for 5 days starting immediately after disease induction) in comparison with untreated controls. Concomitantly, functional assays (phagocytosis, oxidative burst, fluorescence-activated cell sorting analysis of integrin expression) were performed on neutrophils upon in vitro stimulation by IgG-coated latex beads.Results. ALP treatment reduced arthritis incidence and severity and had a protective effect against cartilage and bone erosion. ALP inhibited the conversion of the leukocyte ␤2 integrins into an active conformation upon Fc receptor stimulation of granulocytes. ALP bound to the actin-bundling protein L-plastin and down-modulated filamentous actin assembly in response to stimulation with IgG-coated latex beads in granulocytes. ALP exerted additional inhibitory effects on neutrophil functions associated with cytoskeletal reorganization, such as phagocytosis and oxidative burst.Conclusion. In addition to its antiprotease activity, ALP exerts a variety of blocking effects on neutrophil functions, probably due to modulation of cytoskeletal changes, that may contribute to this inhibitor's antiarthritis potential and qualify it as a multifunctional regulator of inflammatory responses.Antileukoproteinase (ALP), also named secretory leukocyte protease inhibitor for its presence in epithelial secretions and its ability to inhibit neutrophil serine protease, is a highly basic, acid-stable polypeptide. It comprises two mutually homologous highly disulfidebonded domains connected by a short linker (1). ALP circulates in blood, and experimental evidence indicates its physiologic expression by a variety of mucosal epithelial cells, by neutrophils (2), and by murine, but not human, macrophages (3). However, its low molecular weight (11 kd) and its cationic nature (pI Ͼ10) predispose ALP to leaving the circulation for specific accumulation in tissues, especially in cartilage (4,5).In addition to its antiproteolytic properties, ALP has been shown to exert a variety of antiinflammatory actions on macrophages and neutrophils (e.g., the suppression of lipopolysaccharide [LPS]-induced prostaglandin E 2 and matrix metalloproteinase production or

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“…Therefore, two identical chromatographies are performed but a specific modification is introduced between the separations. With this method it would be possible to target defined post-translational modifications [21] like phosphorylations [22,23].…”

Section: Discussionmentioning

confidence: 99%

Multidimensional nano-HPLC for analysis of protein complexes

Wagner

Sickmann

Meyer

et al. 2003

J. Am. Soc. Mass Spectrom.

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The analysis of macromolecular protein complexes is an important factor in understanding most cellular processes, e.g., protein transport into cell organells, signal transduction via biological membranes, apoptosis, energy metabolism, directed motion of cells, and cell division. These complexes are not only built of various numbers of different proteins but also of prosthetic groups and RNA molecules. To understand the role each protein plays in a complex, a complete analysis of all protein compounds is necessary. Therefore, several separation steps have to be coupled to mass spectrometry to identify the proteins. In this work, we describe the application of multidimensional liquid chromatography, SCX-RP-LC as well as SAX-RP-LC, coupled to electrospray ion trap mass spectrometry. Tryptic digested ribosomes were separated by ion exchange chromatography manually collected and prepared for reversed phase chromatography to analyze the peptides via nano-ESI mass spectrometry. The total numbers of identified proteins are compared in consideration of the separation method (SCX-RP versus

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Identification of Modified Proteins by Mass Spectrometry

Cited by 36 publications

References 20 publications

Host cell-specific protein expression in vitro in Ehrlichia ruminantium

Host cell-specific protein expression in vitro in Ehrlichia ruminantium

Antileukoproteinase: Modulation of neutrophil function and therapeutic effects on anti–type II collagen antibody–induced arthritis

Multidimensional nano-HPLC for analysis of protein complexes

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