After nanoelectrospray ionization, the peptides were submitted to tandem mass spectrometry (MS/MS) in the QEXACTIVE Thermo Fisher Scientificc, San Jose, CA) coupled online to the HPLC, followed by detection in the Orbitrap.
Dried samples were resuspended in buffer-A (0.1% formic acid) and loaded onto an Easy-nLC system (Thermo Scientific), coupled online with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific).
In this context, the use of isotope labeling has proven to be a useful strategy for quantitating proteins that are analyzed, using liquid phase separation coupled online to MS/MS sequencing.
To achieve this, we used isobaric labeling (iTRAQ) of peptides, a 10-step HILIC fractionation, and reversed-phase nano-chromatography coupled online with a Q-Exactive Plus mass spectrometer.
Measurements were performed using a nanoflow ultra-high-pressure liquid chromatograph (nano-Advance; Bruker Daltonics, USA) coupled online to an orthogonal quadrupole time-of-flight mass spectrometer (maXis 4G ETD; Bruker Daltonics, USA) via an electrospray ionization source (Captive sprayer; Bruker Daltonics, USA).
Acetylated peptides were then enriched using anti-acetyl-lysine antibodies41 and run on nano liquid chromatography coupled online with tandem mass spectrometry (nanoLC-MS/MS) on an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific), which allows for detection of isotope incorporation into acetylated proteins.
The CDS Pyroprobe 5250 (Chemical Data Systems) pyrolyser was coupled (online) to a 7890A gas chromatography system (Agilent Technologies) and a 5975C mass selective detector (Agilent Technologies).
The pyrolyser (CDS Pyroprobe 5000 series) was coupled online with a 6890 N GC System Gas Chromatograph (Agilent Technologies, Palo Alto, CA, USA) coupled with a 5973 Mass Selective Detector (Agilent Technologies, Palo Alto, CA, USA) single quadrupole mass spectrometer.
Then the supernatant was collected and subjected to nanoelectrospray ionization followed by tandem mass spectrometry (MS/MS) in a Q Exactive (Thermo scientific) coupled online to the HPLC (Dionex) using a C18 column (5 μm, 150Å, Agela Technologies).
The enriched phosphopeptides were analyzed with a Finnigan Surveyor HPLC system coupled online with a LTQ-Oribitrap XL (Thermo Fisher Scientific, Waltham, MA) equipped with a nanospray source.
Due to the complexity of tissue samples, a pre-fractionation of the samples using strong cation exchange (SCX) is typically performed, prior to analytical separation on a nanoscale reverse-phase (RP) column, which is coupled online with the mass spectrometer.
