**6. Integrating NanoLC system and tandem mass spectrometer**

70 Chromatography – The Most Versatile Method of Chemical Analysis

**Figure 3.** Illustration of the flow diagram in online 2D-nanoLC system

loading mode, (b) clean-up mode and (c) analysis mode.

(UltiMateTM/FAMOS/SwitchosTM, LC Packings, Dionex) with the 10-port valve automatic swiching configuration and localization of BioX-SCX column, Trap C18 column and RP C18 column in: (a)

(c)

(a)

(b)

In our example, samples were delivered into the instrument by an automated in-line (integrated LC Parking's System, 5 mm C18 nano-precolumn and 75 μm i.d. × 15 cm column, packed with C18 PepMap100, 100 Å, 3 μm, (LC Parking, Dionex, P/N 160321) via a nanoelectrospray source head and 10 μm inner diameter PicoTip (New Objective, Massachusetts, USA) (Figure 4).

According to the workflow, after 2D-nanoLC separation, peptides were independently analyzed by a QSTAR®XL MS/MS mass spectrometer (Appllied Biosystems/MDS SCIEX, Ontario, Canada) equipped with a nanoESI source. MS and MS/MS spectra were recorded and processed in IDA mode (Information Dependent Acquisition) controlled by Analyst QS software. Typical settings are chosen to select multiply charged ions for MS/MS that produce at least 45-50 ion counts/s in a 0.5 s survey scan. The range of the MS full scan was from 400 to 1200 amu followed by MS/MS fragmentation of the three most intense precursor peptide ions for 1 s each.

**Figure 4.** Setup and demonstration of nanoLC-MS interface, link between nanolC with nanoelectrospray ionization source and tandem mass spectrometry: (a) Schematic diagram of a nanoLC-MS interface; (b) Interface of nanoLC (LC Parkings, Dionex, Netherlands) with QSTAR®XL MS/MS mass spectrometer (Appllied Biosystems/MDS SCIEX, Ontario, Canada). By using this system, complex sample can be injected, desalted, separated and analyzed in complete automatization.
