samdi-tof ms  (SAMDI Tech)

Journal: Bioconjugate chemistry

Article Title: Metabolism of diazirine-modified N -acetylmannosamine analogs to photocrosslinking sialosides

doi: 10.1021/bc2002117

Figure Lengend Snippet: DMB-derivatization of sialic acids reveals that SiaDAz(2me) and SiaDAz(3me) effectively compete with endogenous sialic acid for incorporation into glycoconjugates Jurkat cells were cultured with the indicated compounds, harvested and fractionated. Sialic acids were derivatized by DMB and separated by reverse phase HPLC with fluorescence detection (see Figures S4–S6 ). Peaks were integrated to derive the percentages of Neu5Ac and other sialic acids. Multiple values indicate results from independent experiments. N.D. indicates that a peak corresponding to SiaDAz(4me) could not be identified.

Article Snippet: Membranes were stripped and probed for β–actin to demonstrate equal protein loading in each lane (not shown). (E) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB species derived from cells cultured with Ac 4 ManNAc, incubated with CTxB, and photoirradiated. (F) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB and crosslinked CTxB-GM1a-SiaDAz(2me) derived from cells cultured with Ac 4 ManNDAz(2me), incubated with CTxB, and photoirradiated.

Techniques: Cell Culture, Fluorescence

Journal: Bioconjugate chemistry

Article Title: Metabolism of diazirine-modified N -acetylmannosamine analogs to photocrosslinking sialosides

doi: 10.1021/bc2002117

Figure Lengend Snippet: Jurkat cells were cultured alone or with 1–6. (A) The HPTLC plate was stained for sialic acid using resorcinol. Jurkat cells produce a variety of gangliosides: GM3, GM2, GM1a, and GD1a. The pattern of gangliosides changed upon introduction of unnatural sialic acid precursors Ac4ManNDAz(2me) and Ac4ManNAz, but not other compounds. (B) The HPTLC plate was probed with CTxB-488, which recognizes ganglioside GM1a. Naturally occurring GM1a molecules are present in all ganglioside extracts. Additional species with CTxB-488 reactivity (s✯) appeared when cells were cultured with Ac4ManNDAz(2me), Ac4ManNDAz(3me), or Ac4ManNAz.

Article Snippet: Membranes were stripped and probed for β–actin to demonstrate equal protein loading in each lane (not shown). (E) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB species derived from cells cultured with Ac 4 ManNAc, incubated with CTxB, and photoirradiated. (F) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB and crosslinked CTxB-GM1a-SiaDAz(2me) derived from cells cultured with Ac 4 ManNDAz(2me), incubated with CTxB, and photoirradiated.

Techniques: Cell Culture, High Performance Thin Layer Chromatography, Staining

Journal: Bioconjugate chemistry

Article Title: Metabolism of diazirine-modified N -acetylmannosamine analogs to photocrosslinking sialosides

doi: 10.1021/bc2002117

Figure Lengend Snippet: Jurkat cells were cultured with or without sugar analogs 1–6 for 72 hours and then incubated with CTxB for 45 min at 4 °C. Cells were photoirradiated, lysed, and the lysates separated by reducing SDS-PAGE followed by protein transfer to PVDF membranes. UV-irradiated (A) and non-UV-irradiated samples (B) were probed for the presence of CTxB (+) using an anti-CTxB antibody. A slower mobility band has an apparent molecular weight consistent with formation of a CTxB-GM1a complex (~13 kDa, *). The identity of the very low molecular weight band (<10 kDa) is not known, but it appears to be related to a contaminant in the CTxB preparation. UV-irradiated (C) and non-UV-irradiated samples (D) were probed for the presence of GM1a (++) using CTxB-HRP. A slower mobility band has an apparent molecular weight consistent with formation of a CTxB-GM1a complex (~13 kDa, *). Membranes were stripped and probed for β–actin to demonstrate equal protein loading in each lane (not shown). (E) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB species derived from cells cultured with Ac4ManNAc, incubated with CTxB, and photoirradiated. (F) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB and crosslinked CTxB-GM1a-SiaDAz(2me) derived from cells cultured with Ac4ManNDAz(2me), incubated with CTxB, and photoirradiated.

Article Snippet: Membranes were stripped and probed for β–actin to demonstrate equal protein loading in each lane (not shown). (E) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB species derived from cells cultured with Ac 4 ManNAc, incubated with CTxB, and photoirradiated. (F) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB and crosslinked CTxB-GM1a-SiaDAz(2me) derived from cells cultured with Ac 4 ManNDAz(2me), incubated with CTxB, and photoirradiated.

Techniques: Cell Culture, Incubation, SDS Page, Irradiation, Molecular Weight, Derivative Assay

Journal: Bioconjugate chemistry

Article Title: Metabolism of diazirine-modified N -acetylmannosamine analogs to photocrosslinking sialosides

doi: 10.1021/bc2002117

Figure Lengend Snippet: Jurkat cells were cultured with or without sugar analogs 1–6 for 72 hours and then incubated with CTxB for 45 min at 4 °C. Cells were photoirradiated, lysed, and the lysates separated by reducing SDS-PAGE followed by protein transfer to PVDF membranes. UV-irradiated (A) and non-UV-irradiated samples (B) were probed for the presence of CTxB (+) using an anti-CTxB antibody. A slower mobility band has an apparent molecular weight consistent with formation of a CTxB-GM1a complex (~13 kDa, *). The identity of the very low molecular weight band (<10 kDa) is not known, but it appears to be related to a contaminant in the CTxB preparation. UV-irradiated (C) and non-UV-irradiated samples (D) were probed for the presence of GM1a (++) using CTxB-HRP. A slower mobility band has an apparent molecular weight consistent with formation of a CTxB-GM1a complex (~13 kDa, *). Membranes were stripped and probed for β–actin to demonstrate equal protein loading in each lane (not shown). (E) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB species derived from cells cultured with Ac4ManNAc, incubated with CTxB, and photoirradiated. (F) SAMDI-TOF MS spectrum of SAM-bound monomeric CTxB and crosslinked CTxB-GM1a-SiaDAz(2me) derived from cells cultured with Ac4ManNDAz(2me), incubated with CTxB, and photoirradiated.

Article Snippet: To solidify our assignment of the CTxB-GM1a-SiaDAz(2me) complex, we used self-assembled monolayer (SAM) with matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (denoted SAMDI-TOF-MS) analysis of cell lysates.

Techniques: Cell Culture, Incubation, SDS Page, Irradiation, Molecular Weight, Derivative Assay