Application of displacement chromatography for the analysis of a lipid raft proteome

Trusch, Maria; Böhlick, Alexandra; Hildebrand, Diana; Lichtner, Björn; Bertsch, Andreas; Kohlbacher, Oliver; Bachmann, S.; Schlüter, Hartmut

doi:10.1016/j.jchromb.2009.11.035

Cited by 7 publications

(4 citation statements)

References 35 publications

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“…Details of the HPLC-chip–MS/MS-system used for analysis are described according to Trusch et al [19]. Individual settings of the system are described in the supporting information part “mass spectrometric identification and quantification” (Methods S2) and in Table S1.…”

Section: Methodsmentioning

confidence: 99%

Proteolytic Processing of Angiotensin-I in Human Blood Plasma

et al. 2013

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In mammalian species, except humans, N-terminal processing of the precursor peptide angiotensin I (ANG-1-10) into ANG-2-10 or ANG-3-10 was reported. Here we hypothesize that aminopeptidase-generated angiotensins bearing the same C-terminus as ANG-1-10 are also present in humans. We demonstrate the time dependent generation of ANG-2-10, ANG-3-10, ANG-4-10, ANG-5-10 and ANG-6-10 from the precursor ANG-1-10 by human plasma proteins. The endogenous presence of ANG-4-10, ANG-5-10 and ANG-6-10 in human plasma was confirmed by an immuno-fluorescence assay. Generation of ANG-2-10, ANG-3-10 and ANG-4-10 from ANG-1-10 by immobilized human plasma proteins was sensitive to the cysteine/serine protease inhibitor antipain. The metal ion chelator EDTA inhibited Ang-6-10-generation. Incubation of the substrates ANG-3-10, ANG-4-10 and ANG-5-10 with recombinant aminopeptidase N (APN) resulted in a successive N-terminal processing, finally releasing ANG-6-10 as a stable end product, demonstrating a high similarity concerning the processing pattern of the angiotensin peptides compared to the angiotensin generating activity in plasma. Recombinant ACE-1 hydrolyzed the peptides ANG-2-10, ANG-3-10, ANG-4-10 and ANG-5-10 into ANG-2-8, ANG-3-8, ANG-4-8 and ANG-5-8. Since ANG-2-10 was processed into ANG-2-8, ANG-4-8 and ANG-5-8 by plasma proteases the angiotensin peptides bearing the same C-terminus as ANG-1-10 likely have a precursor function in human plasma. Our results confirm the hypothesis of aminopeptidase mediated processing of ANG-1-10 in humans. We show the existence of an aminopeptidase mediated pathway in humans that bypasses the known ANG-1-8-carboxypeptidase pathway. This expands the knowledge about the known human renin angiotensin system, showing how efficiently the precursor ANG-1-10 is used by nature.

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Section: Methodsmentioning

confidence: 99%

Proteolytic Processing of Angiotensin-I in Human Blood Plasma

et al. 2013

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“…Within a band a high purity of the component is achieved [72]. DE has been shown to be suitable for separation of complex mixture of tryptic peptides [73][74][75] and proteins [76][77][78][79]. One of the characteristics of DE is that DE has a different selectivity compared with GE [77].…”

Section: Separation Of Proteoforms Of Therapeutic Proteins 41 Separamentioning

confidence: 99%

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

Hidayah¹,

Gaikwad²,

Heikaus³

et al. 2020

Proteoforms - Concept and Applications in Medical Sciences

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A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently highquality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.

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“…12,14,23À27 Displacement chromatography has also been shown to be an effective bioanalytical tool for peptide fractionation in both reversed-phase 28À31 and, more recently, for ionexchange systems. 32,33 In addition, elution-modified displacement chromatography in reversed-phase systems has been shown to be efficacious for complex peptide mixtures. 34 To date, protein ion exchange displacement chromatography has not been extensively employed as an analytical tool in the field of proteomics.…”

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confidence: 99%

“…The displacement mode of chromatography offers significant potential for simultaneous concentration and purification of biomolecules. − The concentration of trace impurities in protein ion-exchange displacement separations has been reported . Protein purification in ion exchange systems has been carried out using high molecular weight displacers, − as well as a range of low molecular mass displacers. ,,− Displacement chromatography has also been shown to be an effective bioanalytical tool for peptide fractionation in both reversed-phase − and, more recently, for ion-exchange systems. , In addition, elution-modified displacement chromatography in reversed-phase systems has been shown to be efficacious for complex peptide mixtures …”

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Detection of Trace Proteins in Multicomponent Mixtures Using Displacement Chromatography

2011

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Model protein feed mixtures containing three abundant and seven trace proteins at various concentrations were identified and employed in a series of displacement experiments. Reversed-phase liquid chromatography (RPLC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry were used to evaluate the compositions of both the feed mixtures and effluent fractions from the displacement experiments. The results demonstrated that trace proteins were focused at the boundaries between the abundant solutes where they were enriched and concentrated. For many of the multicomponent feed mixtures, mass spectrometry analyses of the displacement column effluent fractions resulted in the identification of trace proteins that were not detectable in the feed. In addition, the use of minimal or no salt in the carrier solutions enabled the analysis of displacement fractions by direct infusion mass spectrometry. These results are significant in that they indicate that while the presence of abundant proteins can often be problematic for the detection of trace components, displacement chromatography may be able to employ these abundant proteins to focus trace proteins in the displacement train, thus facilitating detection.

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Application of displacement chromatography for the analysis of a lipid raft proteome

Cited by 7 publications

References 35 publications

Proteolytic Processing of Angiotensin-I in Human Blood Plasma

Proteolytic Processing of Angiotensin-I in Human Blood Plasma

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

Detection of Trace Proteins in Multicomponent Mixtures Using Displacement Chromatography

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