Prominence nano hplc system

The protein samples (50 μg each) were acetone-washed, reduced, alkylated and trypsin-digested according to the iTraq protocol (Sciex, Framingham, MA, USA). All labelled samples were combined to make a pooled sample. The peptides were desalted on a Strata-X 33-μm polymeric reversed phase column (Phenomenex, Torrance, CA, USA) and dissolved in a buffer containing 2% acetonitrile and 0.1% formic acid before separation by high pH on an Agilent 1100 high-performance liquid chromatography system (HPLC) using a Zorbax C18 column (2.1 × 150 mm, Agilent Technologies, Palo Alto, CA, USA). The peptides were eluted with a linear gradient of 20 mM ammonium formate, 2% acetonitrile to 20 mM ammonium formate, and 90% acetonitrile at 0.2 ml/min. The 95 fractions were concatenated into 12 fractions and dried. Each fraction was analyzed by electrospray ionization mass spectrometry using the Shimadzu Prominence nano HPLC system (Shimadzu Corporation, Kyoto, Japan) coupled to a 5600 Triple time-of-flight mass spectrometer (Sciex). The peptides were loaded onto a Zorbax 300SB-C18 3.5-μm column (Agilent Technologies) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Two types of samples, + agmatine and control, were used, and 2 × 4 (independent experimental samples) = 8 samples were analyzed simultaneously.

Intracellular oxidized and reduced glutathione levels were quantified as previously described^{43 (link)}. Briefly, a chilled methanol extraction of the whole cells or microsome fractions was freeze-dried and resuspended in 1% acetonitrile, and then applied to a Hydrosphere C18 column (YMC) on a Prominence nano HPLC system (Shimadzu, Kyoto, Japan) in line with a 4000 QTRAP mass spectrometer (AB Sciex Instruments, Foster City, CA) equipped with Hydrosphere C18 column (YMC).

Liquid chromatography and mass spectrometry were performed on a prominence nano-HPLC system (Shimadzu, Tokyo, Japan) coupled with Q-Exactive (Thermo Fisher Scientific, Waltham, MA, USA). The peptides were separated by nano-LC on an in-house packed Ultimate XB-C18 column (3 μm, 12 cm × 75 μm, Welch Materials) at a flow rate of 300 nl/min. Each fraction was dissolved in 0.1% FA, then injected and eluted using a gradient of 5%–30% solvent B (95% acetonitrile, 0.1% FA) over 40 min. The mass spectrometry was operated in data-dependent mode, automatically switching between MS and MS2 acquisition. Survey full-scan MS spectra (m/z 350–1800) were acquired in the Orbitrap with a resolution of 70,000. The 20 most intense ions were sequentially isolated and fragmented by high energy dissociation (HCD). Peptides with unassigned charge states, as well as less than +2, or more than +6, were excluded from fragmentation. Fragment spectra were recorded in the Orbitrap mass analyzer with resolution of 17,500. The dynamic exclusion was enabled with repeat count two, and an exclusion duration 8 s.

Fractions were resuspended in 100 uL of 2% acetonitrile and 0.1% formic acid and loaded onto a Shimadzu Prominence nano HPLC system (Shimadzu, Kyoto, Japan). Peptides were resolved with a gradient of 10–40% acetonitrile (0.1% formic acid) at 300 nL/min over 180 min and eluted through a nanospray interface into a 5600 TripleTOF mass spectrometer (AB Sciex, Framingham, MA). The data was acquired in an information-dependent acquisition mode with Analyst TF 1.6 software (AB Sciex, Framingham, MA). The MS settings were as follows: Ionspray Voltage Floating = 2,300 V, curtain gas = 20, ion source gas 1 = 20, interface heater temperature = 150, and declustering potential = 70 V. The TOF MS scan was performed in the mass range of 400–1,250 Da with a 0.25 s TOF MS accumulation time, whereas the MS/MS product ion scan was performed in the mass range of 100–1,800 Da with a 0.1 s accumulation time. The criteria for product ion fragmentation were set as follows: ions (>400 and <1,250 m/z) with charge states of between 2 and 5 and an abundance threshold of >250 cps. Former target ions were excluded for 10 s after one occurrence. The maximum number of candidate ions per cycle was 20.

In preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Wischgoll et al., 2009), protein samples from mussels receiving NLHS were resolved in 5% SDS polyacrylamide gels by electrophoresis, stained with Biosafe Coomassie (BioRad Laboratories) and destained. Gel slices containing PvHsp70-1 and PvHsp70-2 were excised and freeze-dried overnight. Proteins were then digested with trypsin and the extracted peptides [24 (link)] were loaded onto a C18 column 300SB, 3.5 μm column (Agilent Technologies, USA) and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). The peptides were analyzed by electrospray ionization mass spectrometry with a Shimadzu Prominence Nano HPLC system (Shimadzu, Japan) coupled to a 5600 TripleTOF mass spectrometer (AB Sciex, USA). Protein identification was performed with Mascot sequence matching software (Matrix Science, USA) and the Ludwig NR database.

The desalted peptides were dissolved in a buffer containing 2% acetonitrile 0.1% formic acid before separation by high pH on Q ExactiveTM HF-X using a Zorbax C18 column (2.1 × 150 mm). Peptides were eluted with a linear gradient of 20 mM ammonium formate, 2% ACN to 20 mM ammonium formate, 90% ACN at 0.2 mL/min. The 95 fractions were concatenated into 12 fractions and dried down. Each fraction was analyzed by electrospray ionization mass spectrometry using the Shimadzu Prominence nano HPLC system [Shimadzu] coupled to a 5600 TripleTOF mass spectrometer [Sciex]. Samples were loaded onto an Agilent Zorbax 300SB-C18, 3.5 μm [Agilent Technologies] and separated with a linear gradient of water/acetonitrile/0.1% formic acid (v/v). Fourteen percent of the labeled sample was loaded on the mass spectrometer.

The peptide fractions were loaded onto a nano RP column (5 μm Hypersil C18, 75 μm × 100 mm, Thermo Fisher Scientific Inc., Waltham, MA, USA) mounted in a Prominence Nano HPLC system (Shimadzu, Nakagyo-ku, Kyoto, Japan). The peptides were eluted with an ACN-gradient from 5–40% containing 0.1% formic acid for 65 min at 400 nL/min. The eluates were transferred to Q-Exactive MS (Thermo Fisher Scientific Inc., Waltham, MA, USA ), which was run in positive ion mode and a data-dependent manner with full MS scan from 350–6000 m/z, resolution at 70,000, MS/MS scan with minimum signal threshold 17500, and isolation at 2 Da. To evaluate the performance of the mass spectrometry on iTRAQ-labelled samples two MS/MS acquisition modes, higher collision energy dissociation (HCD) and collision induce dissociation (CID), were employed. To optimize the MS/MS acquisition efficiency of HCD, normalized collision energy (NCE) was systemically examined from 25–70%.