Identification of Fluorescein-5‘-Isothiocyanate-Modification Sites in Proteins by Electrospray-Ionization Mass Spectrometry

Schnaible, Volker; Przybylski, Michael

doi:10.1021/bc990039x

Cited by 45 publications

(40 citation statements)

References 26 publications

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“…An abundant ion corresponding to the mass of the derivatized N-terminal tryptic peptide (FITC-Gly 1 -Lys 9 ) was observed at m/z 611.72 ϩ ), respectively. The observance of these ions is consistent with reports that the fluorescein adduct is labile under collisional activation conditions (38). Although the MS/MS spectrum did not contain a fragment that specifically located the site of conjugation, observance of the FITC-Gly 1 -Lys 9 tryptic peptide provides sufficient evidence to confidently assign the primary labeling site to the N-terminal glycine residue, since modification of Lys 9 would have prevented tryptic digestion at this site (39).…”

Section: Resultssupporting

confidence: 87%

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Kingsbury

Laue

Klimtchuk

et al. 2008

Journal of Biological Chemistry

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Transthyretin (TTR) is normally a stable plasma protein.However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys 10 ) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescencedetected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.

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Section: Resultssupporting

confidence: 87%

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Kingsbury

Laue

Klimtchuk

et al. 2008

Journal of Biological Chemistry

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“…2a) and with 25 mm wide channels separated by 25 mm spacing (Fig. 2b) that were 10 mm deep and the entire assembly was exposed to oxygen plasma for 60 s. To visualize the patterned PLL after plasma exposure, FITC reacted with PLL using a previously published method; 21 bright regions in the fluorescence micrograph indicate FITC-conjugated PLL, and the dark regions correspond to bare glass where PLL film was removed. The degree of PLL etching was estimated by measuring the fluorescence intensity and line width of FITC-conjugated PLL patterns.…”

Section: Resultsmentioning

confidence: 99%

“…Patterned PLL was visualized by conjugating fluorescein isothiocyanate (FITC, Molecular Probes, OR) to PLL via -NH 2 groups. 21 Fluorescence microscopy was used to image FITCconjugated PLL. Sterile bacteriological polystyrene (PS) Petri dish (Fisher, PA) were used as received.…”

Section: Introductionmentioning

confidence: 99%

Patterned cell culture inside microfluidic devices

Rhee¹,

Taylor²,

Tu³

et al. 2005

Lab Chip

263

205

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This paper describes a simple plasma-based dry etching method that enables patterned cell culture inside microfluidic devices by allowing patterning, fluidic bonding and sterilization steps to be carried out in a single step. This plasma-based dry etching method was used to pattern celladhesive and non-adhesive areas on the glass and polystyrene substrates. The patterned substrate was used for selective attachment and growth of human umbilical vein endothelial cells, MDA-MB-231 human breast cancer cells, NIH 3T3 mouse fibroblasts, and primary rat cortical neurons. Finally, we have successfully combined the dry-patterned substrate with a microfluidic device. Patterned primary rat neurons were maintained for up to 6 days inside the microfluidic devices and the neurons' somas and processes were confined to the cell-adhesive region. The method developed in this work offers a convenient way of micropatterning biomaterials for selective attachment of cells on the substrates, and enables culturing of patterned cells inside microfluidic devices for a number of biological research applications where cells need to be exposed to wellcontrolled fluidic microenvironment.

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“…The trypsin digestion of the isolated protein-dye conjugates followed the procedure outlined by Schnaible and Przybylski (1999). Methanol and 3 mM Tris buffer, pH 8.8 were added to bring the lysozyme-dye conjugate concentration to 1 mg/mL and 30% (v/v) methanol.…”

Section: Maldi-tofmentioning

confidence: 99%

“…Since most proteins contain multiple primary amino groups, a number of different products of the labeling reaction differing in the number and location of the attached probes are expected. In order to resolve this uncertainty, several studies have used mass spectrometry to characterize the products of protein labeling reactions and determined the location of the attached probes on the surface of the protein (Grunwaldt et al, 2002;Schnaible and Przybylski, 1999).…”

Section: Introductionmentioning

confidence: 99%

Changes in retention behavior of fluorescently labeled proteins during ion‐exchange chromatography caused by different protein surface labeling positions

Teske

Simon

Niebisch

et al. 2007

Biotech & Bioengineering

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Confocal laser scanning microscopy (CLSM) is a method allowing in situ visualization of protein transport in porous chromatography resins. CLSM requires labeling a protein with a fluorescent probe. Recent work has shown that conjugation of the protein with fluorescent probes can lead to significant changes in the retention time of the protein-dye conjugate with respect to the unlabeled protein. In this study, we show that common labeling procedures result in a heterogeneous mixture of different variants and that attachment location of the fluorescent probe on the protein surface can have a strong effect on the retention of protein-dye conjugate. Lysozyme was labeled with Cy5 and BODIPY-FL succinimidyl esters, followed by chromatographic separation of the different lysozyme-dye conjugates and subsequent determination of the label position using MALDI-TOF-MS. Finally, homogenously labeled lysozyme-dye conjugates were used in CLSM experimentation and compared to published results arising from heterogeneously labeled feedstocks. The results confirm that the attachment location of the fluorescent probe has a strong effect on chromatographic retention behavior. When addressing the binding affinities of the different labeled protein fractions, it was found that native lysozyme was able to displace lysozyme-dye conjugates when the fluorescent label was attached to lysine-33, but not when attached to lysine-97. Finally, it could be shown that when superimposing the single profiles of the three major fractions obtained during a labeling procedure a qualitative picture of the net profile is obtained.

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Identification of Fluorescein-5‘-Isothiocyanate-Modification Sites in Proteins by Electrospray-Ionization Mass Spectrometry

Cited by 45 publications

References 26 publications

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

The Modulation of Transthyretin Tetramer Stability by Cysteine 10 Adducts and the Drug Diflunisal

Patterned cell culture inside microfluidic devices

Changes in retention behavior of fluorescently labeled proteins during ion‐exchange chromatography caused by different protein surface labeling positions

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