1991
DOI: 10.1021/bi00232a030
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Affinity labeling of folate transport proteins with the N-hydroxysuccinimide ester of .gamma.-isomer of fluorescein-methotrexate

Abstract: Fluorescein-methotrexate, a derivative in which the fluorophore is linked via a diaminopentane spacer to either the alpha- or gamma-carboxyl group of the glutamate moiety in the drug [Gapski et al. (1975) J. Med. Chem. 18, 526-528], has been synthesized by an improved procedure and separated by DEAE-Trisacryl chromatography into the alpha- and gamma-isomers (alpha-F-MTX and gamma-F-MTX). Each isomer was characterized by mass spectrometry, elemental analysis, absorbance spectrum, TLC, and reversed-phase HPLC. I… Show more

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Cited by 22 publications
(12 citation statements)
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“…The same behavior of P2 is observed for P3 (data not shown), for which there are unmodified y 3 and modified y 4 *, y 5 *, and y 6 * in ATD Region 1, as well as unmodified y 3 , y 4 , y 5 , and y 6 ions in ATD Region 2. No complete mobility separation was achieved for P3 as well, so that ATD Region 1 corresponds to a highly enriched product ion spectrum of dead-end modification on the second Lys residue, and ATD Region 2, on the first Lys residue.…”
Section: Twim Separation Of Isomeric Dead-end Modified Peptides Formesupporting
confidence: 75%
See 1 more Smart Citation
“…The same behavior of P2 is observed for P3 (data not shown), for which there are unmodified y 3 and modified y 4 *, y 5 *, and y 6 * in ATD Region 1, as well as unmodified y 3 , y 4 , y 5 , and y 6 ions in ATD Region 2. No complete mobility separation was achieved for P3 as well, so that ATD Region 1 corresponds to a highly enriched product ion spectrum of dead-end modification on the second Lys residue, and ATD Region 2, on the first Lys residue.…”
Section: Twim Separation Of Isomeric Dead-end Modified Peptides Formesupporting
confidence: 75%
“…The population of both modified peptide isomers was correlated with the intrinsic reactivities of different Lys residues from reactions conducted at different pH conditions. (J Am Soc Mass Spectrom 2010, 21, 2062-2069) © 2010 Published by Elsevier Inc. on behalf of American Society for Mass Spectrometry C hemical modification of proteins by Nhydroxysuccinimide (NHS) esters is widely used in a series of experiments such as protein purification with biotin-avidin system [1,2], fluorescence labeling [3][4][5], determination of amino acid sidechain solvent accessibility [6 -8], and cross-linking [9,10]. The use of NHS esters for cross-linking strategies has been applied for more than 30 y to identify binding partners, [11] and a more recent approach relies on protein cross-linking coupled to mass spectrometry (MS) experiments to map three-dimensional structures of proteins (MS3D) [12,13].…”
mentioning
confidence: 99%
“…F-MTX binding to RFC was previously documented in L1210 cells, but cellular F-MTX uptake, monitored by fluorimetry, was very slow and largely independent of the RFC (Henderson et al, 1980;Fan et al, 1991). Although no specific interaction between F-MTX and the RFC could be documented in Chinese hamster ovary cells (Assaraf et al, 1989), this may be due to the low sensitivity of the flow cytometry method used (see above).…”
Section: Discussionmentioning
confidence: 93%
“…In the structure shown, the fiunrescein (FITC) is linked via a diaminopentane spacer (DAP) to the ,r-cearboxyl of methotrexate (MTX). Fluorescence microscopy of L. casei spheroplasts labeled with the NHS, ester of F-MTX (from [6] Purification of the folate transport protein from L. casei involves extraction from membranes with Triton X-100, adsorption and elution from microgranular silica, and filtration through Sephadex G-25 [5] The isolated protein has a molecular weight of 18 kDa based upon SDS-PAGE analysis. It is characterized by an unusually high content of hydrophobic amino acids (and methionine) and the absence of carbohydrate.…”
mentioning
confidence: 99%
“…Visualizaion of the L. casei folate transport protein [6] can be accomplished by covalent labeling with a fiuorescein derivative of MTX (F-MTX), whose structure is shown in Fig. 1.…”
mentioning
confidence: 99%