Prestructured MALDI-MS Sample Supports

We report the development of a robust interface for off-line coupling of nano liquid chromatography (LC) to matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) and its application to the analysis of proteolytic digests of proteins, both isolated and in mixtures. The interface makes use of prestructured MALDI sample supports to concentrate the effluent to a small sample plate area and localize the MALDI sample to a predefined array, thereby enriching the analyte molecules and facilitating automated MALDI-MS analysis. Parameters that influence the preparation of MALDI samples from the LC effluent were evaluated with regard to detection sensitivity, spectra quality, and reproducibility of the method. A procedure for data processing is described. The presented nano LC MALDI-MS system allowed the detection of several peptides from a tryptic digest of bovine serum albumin, at analyzed amounts corresponding to one femtomole of the digested protein. For the identification of native proteins isolated from mouse brain by two-dimensional gel electrophoresis, nano LC MALDI-MS increased the number of detected peptides, thereby allowing identification of proteins that could not be identified by direct MALDI-MS analysis. The ability to identify proteins in complex mixtures was evaluated for the analysis of Escherichia coli 50S ribosomal subunit. Out of the 33 expected proteins, 30 were identified by MALDI tandem time of flight fragment ion fingerprinting.

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“…2a. A similar effect was reported by Schuerenberg et al [20] when preparing diluted samples on prestructured sample supports.…”

Section: The Design Of the Nano Lc-maldi Interfacesupporting

confidence: 85%

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: Sample preparation, data analysis, and application to the analysis of complex peptide mixtures

et al. 2005

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“…The gels were stained with colloidal Coomassie blue and the L chains were excised and digested with trypsin after destaining, reduction, and alkylation (38,39). Recovered peptides were prepared for MALDI-TOF mass spectrometry by mixing 1 l of the peptide mixture with 2 l of 30 g/ml ␣-cyano-4-hydroxycinnamic acid and 0.4% trifluoroacetic acid in a 2:1 ethanol:acetone mixture and allowing the droplet to dry on the MALDI sample plate (40). In some cases, the ␣-cyano-4-hydroxycinnamic acid-affinity sample preparation was used to improve sensitivity (41).…”

Section: Mass Spectrometrymentioning

confidence: 99%

Antigen Receptor Editing in Anti-DNA Transitional B Cells Deficient for Surface IgM

Kiefer

Nakajima

Oshinsky

et al. 2008

The Journal of Immunology

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In response to encounter with self-Ag, autoreactive B cells may undergo secondary L chain gene rearrangement (receptor editing) and change the specificity of their Ag receptor. Knowing at what differentiative stage(s) developing B cells undergo receptor editing is important for understanding how self-reactive B cells are regulated. In this study, in mice with Ig transgenes coding for anti-self (DNA) Ab, we report dsDNA breaks indicative of ongoing secondary L chain rearrangement not only in bone marrow cells with a pre-B/B cell phenotype but also in immature/transitional splenic B cells with little or no surface IgM (sIgM−/low). L chain-edited transgenic B cells were detectable in spleen but not bone marrow and were still found to produce Ab specific for DNA (and apoptotic cells), albeit with lower affinity for DNA than the unedited transgenic Ab. We conclude that L chain editing in anti-DNA-transgenic B cells is not only ongoing in bone marrow but also in spleen. Indeed, transfer of sIgM−/low anti-DNA splenic B cells into SCID mice resulted in the appearance of a L chain editor (Vλx) in the serum of engrafted recipients. Finally, we also report evidence for ongoing L chain editing in sIgMlow transitional splenic B cells of wild-type mice.

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“…Mass Spectrometry was performed as following: Protein bands were excised from colloidal Coomassie blue stained gels (Kang et al, 2002) and digested with trypsin after destaining, reduction, and alkylation (Shevchenko et al, 1996;Spodik et al, 2002). Recovered peptides were prepared for MALDI-TOF MS by mixing 1 L of the peptide mixture with 2 l of 30 g/ml a-cyano-4-hydroxycinnamic acid (CHCA), 0.4% trifluoroacetic acid in 2:1 ethanol:acetone mixture and allowing the droplet to dry on the MALDI sample plate (Schuerenberg et al, 2000). In some cases, CHCA affinity sample preparation was used to improve sensitivity (Gobom et al, 2001).…”

Section: Maldi Mass Spectroscopymentioning

confidence: 99%

Cellular nonmuscle myosins NMHC‐IIA and NMHC‐IIB and vertebrate heart looping

Lu¹,

Seeholzer²,

Han³

et al. 2008

Developmental Dynamics

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Flectin, a protein previously described to be expressed in a left-dominant manner in the embryonic chick heart during looping, is a member of the nonmuscle myosin II (NMHC-II) protein class. During looping, both NMHC-IIA and NMHC-IIB are expressed in the mouse heart on embryonic day 9.5. The patterns of localization of NMHC-IIB, rather than NMHC-IIA in the mouse looping heart and in neural crest cells, are equivalent to what we reported previously for flectin. Expression of full-length human NMHC-IIA and -IIB in 10 T1/2 cells demonstrated that flectin antibody recognizes both isoforms. Electron microscopy revealed that flectin antibody localizes in short cardiomyocyte cell processes extending from the basal layer of the cardiomyocytes into the cardiac jelly. Flectin antibody also recognizes stress fibrils in the cardiac jelly in the mouse and chick heart; while NMHC-IIB antibody does not. Abnormally looping hearts of the Nodal ⌬ 600 homozygous mouse embryos show decreased NMHC-IIB expression on both the mRNA and protein levels. These results document the characterization of flectin and extend the importance of NMHC-II and the cytoskeletal actomyosin complex to the mammalian heart and cardiac looping. Developmental Dynamics 237:3577-3590, 2008.

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Prestructured MALDI-MS Sample Supports

Cited by 318 publications

References 36 publications

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: Sample preparation, data analysis, and application to the analysis of complex peptide mixtures

Nanoflow liquid chromatography coupled to matrix-assisted laser desorption/ionization mass spectrometry: Sample preparation, data analysis, and application to the analysis of complex peptide mixtures

Antigen Receptor Editing in Anti-DNA Transitional B Cells Deficient for Surface IgM

Cellular nonmuscle myosins NMHC‐IIA and NMHC‐IIB and vertebrate heart looping

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