Tempo lc maldi spotting system

The serum samples (5μl/subject) from HCC and control groups were pooled (n = 10 subjects/group), diluted thrice, and transferred to a spin filter to remove particles. The highly abundant proteins were discarded with Agilent Human 14 Multiple Affinity Removal System Column (Agilent Technologies, Waldron, Germany). The sample was digested by pancreatin and labeled with 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) reagent kit (Applied Biosystems, CA, USA). The peptides were separated by strong cation exchange (SCX) chromatography through Tempo LC MALDI Spotting system (Applied Biosystems, CA, USA) and processed by 5800 MALDI-Time-of-Flight (TOF)/TOF mass spectrometer (Applied Biosystems, CA, USA). Proteins were identified against the International Protein Index (IPI) database (ipi.HUMAN.V3.62.fasta). A flow chart of iTRAQ-MALDI-MS/MS analysis is shown in Supplemental Figure 2.

All strong cation exchange (SCX) was performed under the following conditions: column size = 150 × 2.1 mm, 5 μm, 200 Å, flow rate = 0.2 ml/min, PolySulfethyl A; PolyLC. Fractions were analyzed on a Tempo^™ LC-MALDI spotting system (Applied Biosystems). Peptides were separated at a flow rate of 2 μl/min, eluted with a 90 min gradient from 8%–40% mobile phase B (98% acetonitrile, 0.1% trifluoroacetic acid) and monitored by UV absorbance at 214 nm. Peptide-containing LC spots were submitted to a 5800 MALDI TOF/TOF^™ analyzer (Applied Biosystems). MS full-scan spectra were acquired from 800–4,000 m/z. Data-dependent tandem MS settings included acquisition of up to 20 of the most intense ion signals per spot. Raw data processing, protein identification, protein relative quantitation and statistical analyses were undertaken with ProteinPilot Software Version 4.0 (Applied Biosystems) against the UniProt database. Protein confidence was set at 95% (equivalent to Unused ProtScore of 1.3). Proteins were accepted with a false discovery rate (FDR) of 1%. Only the proteins identified from three iTRAQ runs with P-values <0.05 (compared with the LADA cohort) and at least two peptides were accepted as unique. Only proteins with a relative expression ratio of ≤0.8- or ≥1.2 between two groups were accepted as significantly down- or upregulated[16 (link), 17 (link)].