Mass spectra were calibrated using the m/z values for two previously identified peptides [2]: APSGFLGMRamide (C. borealis tachykinin-related peptide I [CabTRP I]; m/z = 934.4927) and VYRKPPFNGSIFamide (Val1-SIFamide; m/z = 1423.7845). Peaks for these two peptides were present in the spectra and served as internal calibrants. For SORI-CID experiments, argon was used as the collision gas, the frequency offset was set to −1.8% of the reduced cyclotron frequency

and the voltage amplitude was in the range of 6–8.5 Vbp. SORI-CID spectra were calibrated externally, with a one-point adjustment based upon a [MH−NH3]+ fragment mass. The standards NFDEIDRSGFGA and NFDEIDRSGFG-OMe were characterized by SORI-CID. Mass spectrometric CDK inhibitor analysis was performed using a 6530 quadrupole time-of-flight (Q-TOF) mass analyzer (Agilent Technologies, Santa Clara, CA). Mass spectra (MS and MS/MS) were collected in positive ion mode; the ionization voltage ranged from 1850 to 1950 V and the ion source temperature was held at 350 °C. Spectra

were internally calibrated using methyl stearate (C17H35CO2CH3) and hexakis(1H, 1H, 4H-hexafluorobutyloxy)phosphazine (HP-1221; C24H18O6N3P3F36), check details continuously infused and detected as [M+H]+. CID-MS/MS experiments were executed with precursor ions subjected to CID using nitrogen as the target gas with the collision energy set to 26 V. Chromatographic separation and nano-electrospray ionization (ESI) were performed using a 1260 Chip Cube System (Agilent Technologies) using a ProtID-chip with a 40 nL enrichment column and a 43 or 150 mm × 75 μm analytical column (Agilent Technologies). The enrichment and analytical columns

were packed with 300 Å, 5 μm particles with C18 stationary phase. The mobile phases were 0.1% formic acid/H2O (A) and 0.1% formic acid/acetonitrile (B). Samples (0.2–2 μL) were loaded on the enrichment column using 98:2 (A:B) at 4 μL/min. Tryptic digest samples were analyzed with the 43 mm analytical column using a gradient of 98:2 (A:B) to 20:80 (A:B) over a period of 12 min at 0.6 μL/min. Eyestalk extracts and peptide standards were analyzed with the 150 mm analytical column using a linear gradient of 98:2 (A:B) to 65:35 (A:B) over a period of 5 min, to 40:60 (A:B) at 25 min and 2:98 (A:B) at 35 min using a flow rate of 0.3 μL/min. Calibrated mass spectral this website peak lists were generated using the Omega 8.0 software (IonSpec, Lake Forest, CA, USA). MALDI-FTMS figures were generated using the Boston University Data Analysis software (B.U.D.A.; provided by Dr. Peter O’Connor, University of Warwick, Department of Chemistry, Coventry, England) to produce graphics that were imported into CorelDRAW X4 for final figure production. HPLC Chip–nanoESI Q-TOF MS figures were generated by exporting Mass Hunter (Agilent) chromatograms or mass spectra as metafiles and importing these graphics into CorelDRAW X4 for final figure production. The paired eyestalks of the lobster, H.