Orbitrap xl

Nano LC-MS/MS with collision induced dissociation was performed on an Orbitrap XL (Thermo Fisher, Waltham, MA) integrated with an Eksigent nano-LC. A prepacked reverse-phase column (Acutech Scientific C18) with a dimension of 75 μm × 20 cm containing resin (Biobasic C18, 5-μm particle size, 300-Å pore size, Acutech Scientific, San Diego, CA) was used for peptide chromatography and subsequent CID analyses. ESI conditions using the nano-spray source (Thermo Fisher) for the Orbitrap were set as follows: capillary temperature 220°C, tube lens 110 V and spray voltage of 2.3 kV. The flow rate for reverse-phase chromatography is 0.5 μl/min for loading and 400 nl/min for analytical separation (buffer A: 0.1% formic acid, 3% ACN; buffer B: 0.1% formic acid, 100% ACN). Peptide resolution gradient: 0–40% buffer B over 180 min, then 0% buffer B for 20 min of equilibration. The Orbitrap was operated in data-dependent mode with a full precursor scan at high-resolution (60,000 full width at half maximum, at m/z 400) and 10 MS/MS experiments at low resolution on the linear trap while the full scan was completed. For CID, intensity threshold was 5000, with mass range 350–2000. Spectra were searched using MaxQuant11 in which results with p<0.01 (99% confidence interval) were considered significant and indicating identity.

Fractions were separated on an inline 10 cm × 75 μm I.D. reversed-phase column packed with 5 μm C18 material with 300 Å pore size using a 180 min gradient of 3–32% acetonitrile in 0.1% formic acid. Inline mass spectrometric analysis was performed on an Orbitrap XL (Thermo). Survey scans used a resolving power of 60,000, and the top ten abundant peaks were selected for MS/MS fragmentation and analysis. An exclusion list of 100 members, with early expiration after three readings, and monoisotopic precursor selection (MIPS) were employed.

MS was used to identify lyophilized ABPs using an Orbitrap XL instrument (Thermo Fisher Scientific, Waltham, MA, USA). (A) Formic acid (1%) and (B) CH₃CN (1% formic acid) were used as aqueous mobile phases. At a flow rate 400 L/min for 50 min, a linear gradient of B from 3% to 44% was utilized to elute ABPs. The MS parameters were as follows: 2.2 kV spray voltage, 5 μL/min flow rate, and 350.0–1550.0 m/z mass range. The original peptide sequences from the deer antler (Cervus elaphus) database were identified using the UniProt database.

Isolation of hematinic acid by preparative HPLC was performed as follows: A portion of the lyophilized reaction mixture from condition A (1% NH₃ in methanol, pH 11.5, final conc. 5% aq. H₂O₂, RT) having a crude weight of 3.3 g was purified by preparative reversed-phase HPLC. The method consisted of the following parameters: flow rate was 25 mL/min, mobile phase A consisted of acetonitrile + 0.05% trifluoroacetic acid (TFA), and mobile phase B consisted of water + 0.05% TFA. The run started as follows: 20% A (0–1 min), followed by a gradient that reached 100% A (1–8 min), followed by flushing with 100% A (8–16 min). This produced 41.3 mg (1.3% yield) of hematinic acid, having 93.6% purity. The structure was confirmed by high-resolution electrospray mass spectrometry (Orbitrap XL, Thermo Fisher Scientific) and by nuclear magnetic resonance spectroscopy (NMR; see Supplementary Figures S1–S4 for spectra).

Coomassie Blue stained SDS-PAGE gel bands corresponding to ROR1, ROR2 and CAM-1 ICDs were excised, reduced, cysteine-alkylated, and digested with trypsin in situ[33] (link). Aliquots of the resulting peptides were enriched for phosphopeptides using TiO₂ affinity chromatography (Titansphere; GL Sciences Inc., Torrance, CA). Peptides before and after phospho-enrichment were analyzed by capillary reverse phase liquid chromatography tandem mass spectrometry on an orbitrap mass spectrometer (Orbitrap XL; Thermo Fisher, San Jose, CA). Mass spectra were recorded in data-dependent experiments whereby eight collision-induced dissociation product ion spectra were collected per full MS scan [34] (link). Mass spectra were searched against a database of human proteins using the Mascot program (Matrix Science) and putative phosphorylation sites manually verified.