Table 2. Column Combination Recommendation^a.

precolumn	main column	seq. cov. (%)
Agilent InfinityLab Poroshell 120 SB-C18 2.1 × 5 mm, 1.9 μm	bC1	96
Agilent InfinityLab Poroshell 120 SB-C18 3.0 × 5 mm, 1.9 μm	bC1	97
Agilent InfinityLab Poroshell 120 SB-C18 4.6 × 5 mm, 2.7 μm	bC1	97
Agilent InfinityLab Poroshell 120 SB-C18 4.6 × 30 mm, 2.7 μm	bC1	97
Agilent InfinityLab Poroshell 120 EC-C18 3.0 × 5 mm, 1.9 μm	cC2	93
Agilent AdvanceBio Peptide Mapping 2.1 × 5 mm, 2.7 μm	dC3	96
Agilent AdvanceBio Peptide Mapping 3.0 × 5 mm, 2.7 μm	dC3	96
Agilent AdvanceBio Peptide Mapping 4.6 × 5 mm, 2.7 μm	dC3	96
Waters Atlantis dC18 Column 3.0 μm, 2.1 × 30 mm	eC4	96
Waters ACQUITY UPLC Peptide BEH C18 1.7 μm, 2.1 × 5 mm	eC4	96
Waters ACQUITY UPLC Peptide BEH C18 1.7 μm, 3.0 × 30 mm	eC4	97
Waters ACQUITY UPLC Peptide CSH C18 1.7 μm, 3.0 × 30 mm	fC5	92

^aSequence coverage (seq. cov.) comparison of the main peak CEX fraction of Herceptin (trastuzumab) obtained with the mD-UPLC-MS/MS system with different pre- and main-column combinations. For the analysis, 50 μg of Herceptin (trastuzumab) was injected into the system and the single digestion setup with a trypsin column was used. The data analysis and sequence coverage calculation was accomplished with the PMI-Byos (Byonic) software version 4.0–53 (Protein Metrics Inc.). For the peptide identification, MS/MS spectra were used. The precursor mass tolerance was set to 10 ppm, and a miss cleavage rate of one was permitted.

^bC1: Agilent InfinityLab Poroshell 120 SB-C18 2.1 × 150 mm, 1.9 μm.

^cC2: Agilent InfinityLab Poroshell 120 EC-C18 2.1 × 150 mm, 1.9 μm.

^dC3: Agilent AdvanceBio Peptide Mapping, 2.1 × 150 mm, 2.7 μm.

^eC4: Waters ACQUITY UPLC Peptide BEH C18, 1.7 μm, 2.1 × 150 mm.

^fC5: Waters ACQUITY UPLC Premier Peptide CSH C18, 1.7 μm, 2.1 × 150 mm.