 Off-line 2D nano LC/MS is well suited for extremely complex proteome samples such as whole cell lysates or tissues. It can also be applied to purification and analysis of low abundance proteins or biomarkers from highly complex biological matrices. Here Agilent offers a flexible approach based on the Agilent Nanoflow Proteomics Solution.
 Recommended configuration for an off-line 2D Nano-LC/MS system
The system configuration for off-line 2D LC consists of one LC systems for the 1st dimension and a conventional Agilent 1260 Infinity Nano LC/MS or Agilent 1260 Infinity HPLC-Chip/MS system for the 2nd dimension:
1st dimension LC system - Agilent 1200 Series micro-fraction collection system
- Agilent 1260 Infinity Capillary Pump with micro vacuum degasser (G1382A) - delivers a reliable and robust flow rate necessary for high resolution chromatography
- Agilent 1260 Infinity High Performance Micro Autosampler (G1377A) - for injections from the nL up to the µl range
- Agilent 1260 Infinity Diode-array Detector (G1315B014) with 80-nL flow cell - the 80-nL flow allows for cell high resolution chromatograms and spectra
- Agilent 1260 Infinity Micro Fraction Collector (G1364D) - for fractionation in the low µl range with exact liquid deposition in 96- and 384 well-plates.
2nd dimension LC system
- Agilent 1260 Infinity Nanoflow LC system for MS (G2229A) - Optimum solution to work at stable, precise nanoliter flow rates in combination with nanoelectrospray for highest sensitivity. Includes nanoflow pump, micro vacuum degasser, micro well-plate autosampler, and handheld controller
- Agilent 6500 Series Accurate Mass Q-TOF LC/MS - for highest sensitivity with an extended capacity trap
- Orthogonal nanoelectrospray source (G1982B) - for robust and easy-to-use nanospray
- Nanoflow LC start-up kit (G2228-68700) - includes nano RP and RP enrichment columns and optimized fittings and capillaries that make handling easy
- Agilent 1200 Infinity Series Column Organizer (G1383A) - to adjust valves correctly to minimize the capillary length between the LC system and MS system
- Spectrum Mill MS Proteomics Workbench (G2721A)- for proteomics data analysis
- Thermostat for Agilent 1260 Infinity Autosampler, Well-plate Sampler and Fraction Collector (G1330B) - to prevent degradation of thermolabile samples
- Agilent 1260 Infinity Capillary Pump with micro vacuum degasser (G1382A) - for robust and reliable flow in the µL range. Alternatively, other pumps, e.g. 1200 Series quaternary pump can also be added.
Recommended chromatographic columns
- Ion Exchange column(s) for the 1st dimension of 2D LC
ZORBAX BioSCX Series II, 300 µm × 35 mm, 3.5 µm; strong cation exchanger (5065-9912)
ZORBAX BioSCX Series II, 0.8 mm × 50 mm, 3.5 µm; strong cation exchanger (5065-9942)
- Reversed-phase column for peptide enrichment before 2nd dimension of 2D LC:
ZORBAX 300SB-C18, 300 µm × 5 mm, 5 µm (5/pk) (included in G2228A Nanoflow LC start-up kit) (5065-9913)
- Reversed-phase columns for 2nd dimension of 2D LC:
ZORBAX 300SB-C18, 75 µm × 150 mm, 3.5 µm (included in G2228A Nanoflow LC start-up kit) (5065-9911)
- Holder for 5-mm trap cartridges (5065-9915)
2nd dimension HPLC-Chip/MS system
- Agilent 1260 Infinity Nanoflow LC system for MS (G2229A) - Includes nanoflow pump, micro vacuum degasser, micro well-plate autosampler, and handheld controller. Optimum solution to work at stable, precise nanoliter flow rates in combination with nanoelectrospray for the highest sensitivity in your proteomics work
- Agilent 1260 Infinity HPLC-Chip/MS system (G4240A) - fully integrated HPLC-Chip offers easy to use, robust nanospray LC-MS for proteomics applications.
- Agilent 1260 Infinity Capillary Pump with micro vacuum degasser (G1382A) - for robust and reliable flow in the µL range.
- Agilent 6500 Series Accurate Mass Q-TOF LC/MS - offers highest sensitivity with an extended capacity trap
- Spectrum Mill MS Proteomics Workbench (G2721A)- for proteomics data analysis
- Thermostat for Agilent 1260 Infinity Autosampler, Well-plate Sampler and Fraction Collector (G1330B) - to prevent degradation of thermolabile samples
Recommended HPLC-Chips and chromatographic columns
- Ion Exchange column(s) for the 1st dimension of 2D LC
ZORBAX BioSCX Series II, 300 µm × 35 mm, 3.5 µm; strong cation exchanger (5065-9912)
ZORBAX BioSCX Series II, 0.8 mm × 50 mm, 3.5 µm; strong cation exchanger (5065-9942)
- Protein ID #1 HPLC-Chip - for low-medium complexity samples. Integrated enrichment column, separation column, capillaries and spray emitter directly on the chip. 40nl enrichment column and 43 mm x 75um analytical column packed with ZORBAX 300SB C-18 5um. (G4240-62001), ZORBAX 300SB-C18, 300 µm × 5 mm, 5 µm (5/pk) (included in G2228A Nanoflow LC start-up kit) (5065-9913)
- Protein ID #2 HPLC-Chip - for medium-high complexity samples. Integrated enrichment column, separation column, capillaries and spray emitter directly on the chip. 40-nl enrichment column and 150 mm x 75-µm analytical column packed with ZORBAX 300SB C-18 5 µm (G4240-62002)
Options for both conventional Nano LC and HPLC-Chip/MS systems
- Thermostats (G1330B) for micro fractions collector and/or high performance micro autosampler - to prevent degradation of thermolabile samples
- Micro fraction collector (G1364D) and 2-position/6-port valve (G1157A) - for capacity extension (G1364D)
- 100-µL flow sensor - for flow rates up to 100 µL/min (G1376-60002)
Back to Multidimensional LC Separation of Protein Digests
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