Page 19 - Shimadzu Journal vol.9 Issue2

Biopharmaceutical

100 µg of trastuzumab control and stress induced samples were Table 1. Details of analytical conditions for bottom-up approach
incubated with reduction buffer containing 8 M urea and 5 mM HPLC system Nexera™ X2
dithiothreitol at 37°C for 60 mins. Solutions were then alkylated Shim-pack™ Arata Peptide C18
Column (100 mm × 2.0 mmI.D., 2.2 µm)
with 20 mM of iodoacetamide at room temperature for 30 mins. (P/N: 227-32806-02)
At the end of incubation period, 50 mM Tris-HCl buffer (pH 8) Column oven 40°C
A-0.1% formic acid in water
was added to the solutions. Finally, trypsin was added in 1: 50 ratio Mobile phases B-0.1% formic acid in acetonitrile
and overnight digestion was carried out. Reaction was quenched Flow rate 0.3 mL/min
with diluted formic acid and desalting was carried out using Solid 0-3 min → 1 (%); 3-45 min → 1-30 (%);
Gradient program (B%) 45-46 min → 30 (%); 46-46.1 min→ 30-90 (%);
Phase Extraction (SPE) cartridges. Eluent obtained at the end of 46.1-50 min →90 (%); 50-50.1 min→ 1 (%);
60 min → stop
SPE clean-up was evaporated using vacuum centrifugation and re- LCMS system LCMS-9030
constituted in water: formic acid: acetonitrile (100:1:2 v/v) solution Interface Heated ESI
and analysed with LCMS-9030 (shown in Fig. 1) for peptide map- Polarity Positive
ping analysis. Mobile phase consisting of 0.1% formic acid in water Acquisition mode DDA
and 0.1% formic acid in acetonitrile was used on Shim-pack™ Arata Mass range for TOF survey scan 200-2500 m/z
Peptide C18 column for the chromatographic separation. Analysis Mass range for precursor ion 220-2000 m/z
was performed in Data Dependent Acquisition (DDA) mode in
Mass range for MS/MS scan 100-2800 m/z
positive polarity using Electro Spray Ionization (ESI) interface .
3
Collision energy spread 18-52 V
DDA data acquisition was controlled by the LabSolutions™ LCMS Interface: 300°C
Temperatures Desolvation line: 200°C
software. Mass range of 200-2500 m/z was used for MS1 TOF Heater block: 400°C
survey scan. Base peak chromatogram intensity threshold of more Heating gas: 15 L/min
Gas flow rates Nebulizing gas: 3 L/min
than 1000 was used to trigger the MS/MS fragmentation with col- Drying gas: 15 L/min
lision energy spread of 18-52 V. Use of collision energy spread al-
lowed acquisition of comprehensive fragmentation pattern for any
given precursor ion. Seven dependent (MS/MS) events were set
to allow sufficient MS/MS data collection. Mass range of 100 to
2800 m/z was used to obtain MS/MS spectra. Ion exclusion and
inclusion settings are available in the LabSolutions LCMS software
to automatically exclude background ions and include ions of in-
terest, respectively.
All data acquisition was performed with single external TOF
calibration. No intermediate TOF calibration/lock masses were
used during the data acquisition/processing. Details of analytical
conditions are given in Table 1.
The LCMS-9030 quadrupole time-of-flight (Q-TOF) mass
spectrometer is a powerful instrument that integrates the world’s
fastest and most sensitive quadrupole technology with TOF capa-
bilities for accurate mass measurement. Patented technologies of UFaccumulation™ UFgrating™
LCMS-9030, UF-FlightTube™ and iRefTOF™, ensure excellent UF-FlightTube™ iRefTOF™
Mass Measurement Accuracy (MMA) with stability which helps in
Fig. 1. LCMS -9030 Quadrupole Time of Flight Mass Spectrometer
TM
identification of different peptides and PTMs present in the sample.
UFaccumulation™ and UFgrating™ offer superior sensitivity which
helps in detecting low abundant PTMs present in the samples.
DDA data acquired by LCMS-9030 was processed using ‘Protein was considered as fixed. Other PTMs like oxidation, deamidation,
Metrics’ software suite . Settings of precursor mass tolerance of ‘6 Gln->pyro-Glu, Glu->pyro-Glu, ammonia-loss/ succinamide for-
4
ppm’ and fragment mass tolerance of ‘20 ppm’; maximum 2 missed mation, dioxidation, dethiomethylation were considered as vari-
cleavages; and fully specific trypsin digestion efficiency were used able. N-glycan 52 common biantennary database present in the
for peptide/PTMs identification. Carbamidomethyl modification software was used to obtain information about glycosylation.

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