**5. Two-dimensional nano liquid chromatography (2D-nanoLC)**

The basic setup of an online two-dimensional nano liquid chromatography (2D-nanoLC) system (LC Parking, Dionex, The Netherlands) was developed for improved separation and hydrophobic peptide recovery, especially for complex peptides made from enzymatic digests of selected proteomes. The system works with the principle of elution of the digested peptides from the first dimension SCX column with injected salt solution plugs of increasing concentration. The eluted peptides are again trapped and introduced into the nanoflow path for separation and analysis by second dimension RP column and tandem mass spectrometry. The great advantage of the system is a robust and fully automated separation. The methods are easy to set up and composed of identical runs differing only in the concentration of injected salt plugs.

For the mentioned above online 2D-nanoLC system, the following columns were used: (i) strong cation exchange (SCX) column (500 μm i.d.×1.5 cm) packed with BioX-SCX, 300 Å, 5 μm, (LC Parking, Dionex, P/N 161395); (ii) Trap column: 300 μm i.d.× 0.5 cm, packed with PepMapTM C18, 100 Å, 5 μm, (LC Parking, Dionex, P/N 160454); (iii) Reversed phase (RP) column: 75 μm i.d.× 15 cm, packed with C18 PepMap100, 100 Å, 3 μm, (LC Parking, Dionex, P/N 160321). The column physico-chemical properties, functions, and the mobile phase, loading/eluted solvents for the flow diagram in online 2D-nanoLC system (UltiMateTM/FAMOS/SwitchosTM, LC Packings, Dionex) with the 10-port valve automatic swiching configuration are shown in details in table 1 and figure 3.

covered with trypsin buffer (typically, 50 μl or more).

**4.3. Sample cleanup with C-18 ZipTips** 

contingency.

concentration of injected salt plugs.

in table 1 and figure 3.

were then reduced by incubating with 5 mM DTT solution at 56oC for 45 min and alkylated for 1 hr with 20 mM IAA solution in darkness at room temperature. The membrane proteins were digested by adding trypsin buffer (0.03 μg/μl in 10 mM ammonium bicarbonate containing 10% (vol/vol) acetonitrile) and incubating overnight at 37oC. Check if all solution was absorbed and add more trypsin buffer, if necessary. Gel pieces should be completely

The resulting peptide digestion products were extracted by adding 100 μl of extraction buffer (1:2 (vol/vol) 5% formic acid/acetonitrile) to each tube and incubated for 15 min at 37 °C in a shaker. All extracts were saved and dried and re-dissolved in 10–20 μl of 0.1% FA, incubated for 2–5 min in the sonication bath and centrifuged for 15 min at 10,000 rpm at the bench-top centrifuge. The obtained supernatant was applied for binding the samples onto micro pipette tips (μC18), catalog number ZTC18S096 (ZipTip®, Millipore Co., Billerica, MA 01821 USA), equilibrated by being aspirated and dispensed with 100% acetonitrile, 40% acetonitrile/0.1% FA, 0.1% FA solutions. The samples were washed (4 times by aspirating and dispensing) with 15 μl of 0.1% FA), then eluted with 10 μl of 40% acetonitrile/0.1% FA. Appropriate aliquots were withdrawn for LC-MS/MS analysis or store at −20°C as

**5. Two-dimensional nano liquid chromatography (2D-nanoLC)** 

The basic setup of an online two-dimensional nano liquid chromatography (2D-nanoLC) system (LC Parking, Dionex, The Netherlands) was developed for improved separation and hydrophobic peptide recovery, especially for complex peptides made from enzymatic digests of selected proteomes. The system works with the principle of elution of the digested peptides from the first dimension SCX column with injected salt solution plugs of increasing concentration. The eluted peptides are again trapped and introduced into the nanoflow path for separation and analysis by second dimension RP column and tandem mass spectrometry. The great advantage of the system is a robust and fully automated separation. The methods are easy to set up and composed of identical runs differing only in the

For the mentioned above online 2D-nanoLC system, the following columns were used: (i) strong cation exchange (SCX) column (500 μm i.d.×1.5 cm) packed with BioX-SCX, 300 Å, 5 μm, (LC Parking, Dionex, P/N 161395); (ii) Trap column: 300 μm i.d.× 0.5 cm, packed with PepMapTM C18, 100 Å, 5 μm, (LC Parking, Dionex, P/N 160454); (iii) Reversed phase (RP) column: 75 μm i.d.× 15 cm, packed with C18 PepMap100, 100 Å, 3 μm, (LC Parking, Dionex, P/N 160321). The column physico-chemical properties, functions, and the mobile phase, loading/eluted solvents for the flow diagram in online 2D-nanoLC system (UltiMateTM/FAMOS/SwitchosTM, LC Packings, Dionex) with the 10-port valve automatic swiching configuration are shown in details


**Table 1.** The type of columns with their physico-chemical properties, functions and the mobile phase, loading/eluted solvents that were used for basic experimental setup of an online two-dimensional nano liquid chromatography system (2D-nanoLC, UltiMateTM/FAMOS/SwitchosTM, LC Parking, Dionex, The Netherlands).

For in-gel digest samples, as the rst step, the peptide mixture was re-dissolved in 30 μl of 0.1% FA and directly loaded onto a strong cation exchange (SCX) column (500 μm i.d.×1.5 cm, 5 μm, 300 Å) at a ow rate of 30 μl/min. Bound peptides were eluted by following ammonium acetate gradients from 10 mM to 2 M: 10 mM, 20 mM, 40mM, 60mM, 80mM, 100 mM, 200 mM, 500mM, 1M and 2M and then desalted and concentrated independently on a C18 trap column (300 μm i.d. ×0.5 cm, 5 μm, 100 Å). The eluted peptides were further separated onto a reversed phase C18 column (75 μm i.d.×15 cm, 5 μm, 100 Å), for the second dimension. The ow rate was maintained at 200 nl/min with solvent A (0.1% FA in LC-MS grade water). With 10 different concentrations of ammonium acetate (plugs), there should be 10 identical runs.

2D-NanoLC-ESI-MS/MS for Separation and Identification of Mouse Brain Membrane Proteins 71

After washing (~12 min), peptides were eluted from a reversed phase C18 column using the solvent B (0.1% FA in 85% LC-MS grade ACN) gradients: from 5 to 20% of solvent B in 25 min, 20 to 70% in 28 min, 70 to 100% in 10 min and maintaining 100% B in 10 min, and back

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,

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

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

**Figure 4.** Setup and demonstration of nanoLC-MS interface, link between nanolC with

sample can be injected, desalted, separated and analyzed in complete automatization.

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

(a) (b)

to 5% B in 5 min.

Massachusetts, USA) (Figure 4).

peptide ions for 1 s each.

**Figure 3.** Illustration of the flow diagram in online 2D-nanoLC system (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) loading mode, (b) clean-up mode and (c) analysis mode.

After washing (~12 min), peptides were eluted from a reversed phase C18 column using the solvent B (0.1% FA in 85% LC-MS grade ACN) gradients: from 5 to 20% of solvent B in 25 min, 20 to 70% in 28 min, 70 to 100% in 10 min and maintaining 100% B in 10 min, and back to 5% B in 5 min.
