Yang et al. 10.1073/pnas.0708574104. |
Fig. 5. Alignment of Kv1.2 aa sequences. Peptides identified by MS are highlighted in yellow, those identified phosphorylation sites are in red, those forming transmembrane segments S1-S6 are underscored, and positions that differ between rat, mouse, and human Kv1.2 are boxed.
Fig. 6. Identification of phosphorylation sites on Kv1.2 in rat brain (A-C) and heterologous HEK293 cells (D) using LC-MS/MS. (A) The spectrum of a doubly charged, singly phosphorylated Kv1.2 peptide obtained from LC-MS/MS at m/z 1,089.13 that was fragmented to produce a MS/MS spectrum with y- and b-ion series that described the sequence ETEGEEQAQYLQVTpSCPK (amino acids 420-437). The phosphorylation site was unambiguously assigned to S434 due to mass assignments from b-eliminated y4 and b15 fragment ions with neutral loss of phosphoric acid H3PO4. (B) The spectrum of a doubly charged, singly phosphorylated Kv1.2 peptide obtained from LC-MS/MS at m/z 532.75 that was fragmented to produce a tandem mass spectrum with y- and b-ion series that described the sequence IPpSSPDLKK (amino acids 438-446). The phosphorylation site was unambiguously assigned to S440 due to mass assignments from b-eliminated b3 and y7 fragment ions and neutral loss of phosphoric acid H3PO4. (C) The spectrum of a singly charged, singly phosphorylated Kv1.2 peptide at m/z 1,064.35 that was fragmented to produce a tandem mass spectrum with y-ion and b-ion series that described the sequence IPSpSPDLKK (amino acids 438-446). The phosphorylation site was unambiguously assigned to S441 due to observe neutral loss of phosphoric acid H3PO4 at m/z 965.35 and mass assignments from beta-eliminated b4, b5, b6, b7, b8 and y6, y7 and y8 fragment ions. (D) The spectrum of a doubly charged, singly phosphorylated Kv1.2 peptide at m/z 508.75 from heterologous HEK293 cells that was fragmented to produce a tandem mass spectrum with y- and b-ion series that described the sequence SRpSASTISK (amino acids 447-455). The phosphorylation site was unambiguously assigned to S449 due to mass assignments from b-eliminated y7, y8, and b3 fragment ions with neutral loss of phosphoric acid H3PO4.
Fig. 7. Mass spectrum of a doubly charged, doubly phosphorylated peptide with the sequence IPpSpSPDLKK as shown in Fig. 1. (Insets) Higher resolution views of low-abundance peaks.
SI Materials and Methods
In-Gel Digestion.
The Kv1.2 band was directly excised from SDS/PAGE gels, destained with 50% acetonitrile in 25 mM ammonium bicarbonate, and dried in a speed-vacuum concentrator. After reduction and alkylation of Cys, gel pieces were washed and dehydrated. Dried gel pieces were swollen with 25 mM ammonium bicarbonate (pH 8.0) containing 10 ng/ml trypsin (Promega) and incubated for 18 h at 37°C. Tryptic-digested peptide mixtures released into the supernatant were extracted with 50% acetonitrile in 5% formic acid and dried in a speed vacuum concentrator.MS-LC-MS/MS.
Protein identification was performed using a UPLC system (nanoACQUITY; Waters) coupled to an LTQ-FT ion trap MS (Thermo-Fisher) through a Microm (Microm BioResourses) "Plug and Play" vacuum spray source. This instrument has an increased mass accuracy and is more sensitive than the LCQ MS used in our previous studies. Peptides were loaded on to a Waters Symmetry C18 280 mm ´ 20 mm nanoAcquity trap column at a loading flow rate of 15 ml/min. Peptides were then eluted from the trap and separated by a Waters 100 mm ´ 100 mm UPLC column using a 90 min gradient of 2-80% buffer B (Buffer A, 0.1% formic acid; Buffer B, 95% acetonitrile, and 0.1% formic acid). The top four ions in each survey scan were subjected to automatic low energy CID. MS/MS spectra were interpreted with the Mascot search engine (Matrix Science). Database searches, through Mascot, were performed with a mass tolerance of 20 ppm, MS/MS tolerance of 0.4 Da and one missing cleavage site and carbamidomethylation of cysteine, oxidation of Met and phosphorylation on Ser, Thr and Tyr residues were allowed. Each filtered MS/MS spectra exhibiting possible phosphorylation was manually checked and validated. Existence of a 98 Da mass loss (-H3PO4: phosphopeptide specific CID neutral loss) and any ambiguity of phosphorylation sites were carefully examined.Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC).
HEK293 cells transiently coexpressing Kv1.2 and Kvb2 were grown for at least five cell divisions in either normal arginine (12C6,14N4; "Arg-0") and lysine (12C6,14N2; "Lys-0") media, or in isotopic Arg (13C6,14N4; "Arg-6") and Lys (13C6,15N2; "Lys-8") labeling media, before harvest. After labeling, cells were solubilized in lysis buffer containing 1% Triton X-100, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1 mM sodium orthovanadate, 5 mM NaF, 5 mM sodium pyrophosphate, 1 mM PMSF, and a protease inhibitor mixture. The lysates were centrifuged at 12,000 ´ g and small scale (1 ml) immunoprecipitation performed. The cell lysates were incubated overnight with affinity purified rabbit anti-Kv1.2C polyclonal Ab and subsequently incubated with protein G beads for 2 h at 4°C. After six washes with lysis buffer, bound proteins were eluted by boiling in SDS sample buffer for 5 min. An aliquot of the immunoprecipitation reaction was analyzed by immunoblot to estimate equal levels of Kv1.2 expression in the isotope and non-isotope-labeled samples. The remainder was subjected to size fractionation by SDS-PAGE gel electrophoresis, Coomassie staining, and separate excision of upper and lower Mr forms of Kv1.2 from both samples. Portions of excised bands were mixed in various combinations, subjected to in-gel digestion followed by LC-MS/MS as described above. After database searches as described above, relative spectral intensities were determined using Xcalibur (version 2.0) software.