This paper introduces a new multidimensional separation technique to reduce the complexity of complex serum samples. The technique uses a high-capacity multiple affinity removal column to specifically remove six high-abundance proteins (albumin, IgG, transferrin, haptoglobin, IgA, and anti-trypsin), and then uses a macro-porous reverse-phase C18 (mRP-C18) column to fractionate the depleted serum. Under the optimized mRP-HPLC condition, protein recoveries for depleted serum samples were greater than 99%. Reproducibility among patient samples was excellent based on the UV trace overlays. The mRP-C18 fractionated serum samples were further analyzed by 1D-PAGE and 2D-LC-MS/MS, the results showed that the technique can successfully reduce the complexity of serum samples and improve the detection sensitivity of low-abundance proteins.
The study applies a new multidimensional separation technique to reduce sample complexity in the comparative analysis of human serum from breast cancer patients and healthy controls. The method involves removing high-abundance proteins followed by fractionation to identify potential biomarkers.
A High Capacity Multiple Affinity Removal Column (Hu6-HC) was used to remove six specific high-abundant proteins: albumin, IgG, transferrin, haptoglobin, IgA, and anti-trypsin. The depleted serum was then fractionated using a macro-porous reverse-phase C18 (mRP-C18) column on an Agilent 1100 HPLC system.
| Affinity Column Dimensions | 4.6-mm id x 50 mm |
|---|---|
| Affinity Column Part Number | 5188-5332 |
| mRP-C18 Column Dimensions | 9.4-mm id x 50 mm |
| mRP-C18 Column Part Number | 5188-5345 |
| In-gel Digestion Kit Part Number | 5188-2749 |
| HPLC System Model | Agilent 1100 HPLC |
| MS/MS Trap Column Dimensions | 0.3-mm id x 5 mm |
| MS/MS Reverse-Phase Column Dimensions | 0.075-mm id x 150 mm |
| MS/MS Detector Model | MSD Trap XCT |
The method specifically removes six high-abundant proteins: albumin, IgG, transferrin, haptoglobin, IgA, and anti-trypsin.
An Agilent High Capacity Multiple Affinity Removal Column (Hu6-HC) is used.