Offline two-dimensional chromatography is a common means to achieve deep proteome coverage. To reduce sample complexity and dynamic range and to utilize mass spectrometer (MS) time efficiently, high chromatographic resolution of and good orthogonality between the two dimensions is needed. Ion exchange and high pH reversed phase chromatography are often used for this purpose. However, the former requires desalting to be MS-compatible and the latter requires fraction pooling to create orthogonality. Here, we report an alternative first-dimension separation technique using a commercial trimodal phase incorporating polar embedded reversed phase, weak anion exchange and strong cation exchange material. The column is capable of retaining polar and nonpolar peptides alike without noticeable breakthrough. It allows separating ordinary and TMT-labelled peptides under mild acidic conditions using an acetonitrile gradient. The direct MS compatibility of solvents and good orthogonality to online coupled C18 columns enable a straightforward workflow without fraction pooling and desalting while showing comparable performance to the other techniques. The method scales from low to high microgram sample quantity and is amenable to full automation. To demonstrate practical utility, we analyzed the proteomes of ten human pancreatic cancer cell lines to a depth of >8,700 quantified proteins.

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