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Medientyp:
E-Artikel
Titel:
Mass spectrometric characterization of recombinant rat 5‐hydroxytryptamine receptor 1A (5‐HT1AR) expressed in tsA201 human embryonic kidney cells
Beteiligte:
Heo, Seok;
Yang, Jae‐Won;
Huber, Marie L.;
Planyavsky, Melanie;
Bennett, Keiryn L.;
Lubec, Gert
Beschreibung:
<jats:p>The 5‐hydroxytryptamine 1<jats:styled-content style="fixed-case">A</jats:styled-content> receptor (serotonin 1<jats:styled-content style="fixed-case">A</jats:styled-content> receptor; 5‐<jats:styled-content style="fixed-case">HT</jats:styled-content><jats:sub>1<jats:styled-content style="fixed-case">A</jats:styled-content></jats:sub><jats:styled-content style="fixed-case">R</jats:styled-content>) is involved in a large series of brain functions, and roles in anxiety, depression, and cognition have been reported. So far, published information on mass spectrometrical characterization of 5‐<jats:styled-content style="fixed-case">HT</jats:styled-content><jats:sub>1A</jats:sub>R is limited to the presence of two 5‐<jats:styled-content style="fixed-case">HT</jats:styled-content><jats:sub>1<jats:styled-content style="fixed-case">A</jats:styled-content></jats:sub><jats:styled-content style="fixed-case">R</jats:styled-content> peptides in rat's whole brain as observed by in‐solution digestion followed by <jats:styled-content style="fixed-case">LC</jats:styled-content>‐<jats:styled-content style="fixed-case">MS</jats:styled-content>/<jats:styled-content style="fixed-case">MS</jats:styled-content>. Knowledge about the protein sequence and <jats:styled-content style="fixed-case">PTM</jats:styled-content>s, however, would have implications for generation of specific antibodies and designing studies on the 5‐<jats:styled-content style="fixed-case">HT</jats:styled-content><jats:sub>1<jats:styled-content style="fixed-case">A</jats:styled-content></jats:sub><jats:styled-content style="fixed-case">R</jats:styled-content> at the protein level. A rat recombinant 5‐<jats:styled-content style="fixed-case">HT</jats:styled-content><jats:sub>1<jats:styled-content style="fixed-case">A</jats:styled-content></jats:sub><jats:styled-content style="fixed-case">R</jats:styled-content> was extracted from the ts<jats:styled-content style="fixed-case">A</jats:styled-content>201 cell line, run using several gel‐based principles with subsequent in‐gel digestion with several proteases, chymotrypsin, trypsin, <jats:styled-content style="fixed-case">A</jats:styled-content>sp<jats:styled-content style="fixed-case">N</jats:styled-content>, proteinase <jats:styled-content style="fixed-case">K,</jats:styled-content> and pepsin followed by nano‐<jats:styled-content style="fixed-case">LC</jats:styled-content>‐<jats:styled-content style="fixed-case">ESI</jats:styled-content>‐<jats:styled-content style="fixed-case">MS</jats:styled-content>/<jats:styled-content style="fixed-case">MS</jats:styled-content> analysis on a high capacity ion trap and an <jats:styled-content style="fixed-case">LTQ O</jats:styled-content>rbitrap <jats:styled-content style="fixed-case">V</jats:styled-content>elos. Using two search engines, <jats:styled-content style="fixed-case">M</jats:styled-content>ascot and <jats:styled-content style="fixed-case">M</jats:styled-content>odiro™, the recombinant 5‐<jats:styled-content style="fixed-case">HT</jats:styled-content><jats:sub>1<jats:styled-content style="fixed-case">A</jats:styled-content></jats:sub><jats:styled-content style="fixed-case">R</jats:styled-content> was identified showing 94.55% sequence coverage. A single phosphorylation at <jats:styled-content style="fixed-case">S</jats:styled-content>301 was identified and verified by phosphatase treatment and a series of amino acid substitutions were detected. Characterization of 5‐<jats:styled-content style="fixed-case">HT</jats:styled-content><jats:sub>1<jats:styled-content style="fixed-case">A</jats:styled-content></jats:sub><jats:styled-content style="fixed-case">R</jats:styled-content>, a key player of brain functions and neurotransmission, was shown and may enable generation of specific antibodies, design of future, and interpretation of previous studies in the rat at the protein level.</jats:p>