Abstract
Nano HPLC-MS/MS separation and detection of peptides for proteomic analysis is usually performed upon tryptic digest of proteins and peptide pre-concentration on trap columns. Pre-concentration on trap columns is needed for sample wash (removal of salts and impurities), sample focusing prior to separation, and volume reduction. Usually, trap columns are mounted on selection valves close to the separation column in order to keep the void volume low and to enable injection of large sample amounts onto nano-separation column. Since separation columns are operated at elevated temperature of ≥45 °C and they are mounted on the same valve as the trap column (in the column oven); loading samples at elevated temperature will result with significant loss of analytes. A method for loading samples on a trap column at 60 °C was developed and optimized. No sample loss was observed when the optimized method was used for analysis of standards and of complex biological samples.
Highlights
Nano HPLC and mass spectrometry (MS) are analytical methods of choice for separation and detection of peptides generated through enzymatic digestion of proteins
Separation of peptides for proteomics analysis is performed using nano HPLC columns with inner diameters (ID) of 50 μm to 100 μm. These small ID will contribute to increased sensitivity of the separation and detection method, which is electrospray ionization—mass spectrometry (ESI-MS) but will limit the amount of sample in terms of volume that can be injected [7] on a nano-separation column
∆H0 is the enthalpy of the retention and ∆S0 is the entropy of the retention at the given temperature T, R is the universal gas constant, and φ the phase ration
Summary
Nano HPLC and mass spectrometry (MS) are analytical methods of choice for separation and detection of peptides generated through enzymatic digestion of proteins. Separation of peptides for proteomics analysis is performed using nano HPLC columns with inner diameters (ID) of 50 μm to 100 μm These small ID will contribute to increased sensitivity of the separation and detection method, which is electrospray ionization—mass spectrometry (ESI-MS) but will limit the amount of sample in terms of volume that can be injected [7] on a nano-separation column. Columns, the trapping column and the separation column are operated at elevated temperature but the loading mobile phase is cooled and the cooling effect applies only to the trapping column and does not directly influence the separation performance. Trap columns are always mounted on the same switching valve as the separation column, Figure 1 This approach is needed in order to minimize the void volume and enable fast sample transfer from the trap column to the separation column and avoid additional separation on the trap column.
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