This Application Note illustrates the potential of combining protein depletion using the Agilent Multiple Affinity Removal System and protein or peptide fractionation using the Agilent 3100 OFFGEL Fractionator for the LC-MS based analysis of plasma proteins.
The document illustrates a method for comprehensive analysis of complex protein mixtures, specifically human plasma. It details how combining immuno-depletion of abundant proteins with isoelectric point (pI) based fractionation via the Agilent 3100 OFFGEL Fractionator improves detection of low-abundance proteins compared to standard methods.
Two experimental setups are described: direct protein OFFGEL electrophoresis followed by digestion and analysis, or initial digestion followed by peptide OFFGEL electrophoresis. The text provides specific protocols for sample preparation, including reconstitution, depletion using the Multiple Affinity Removal System, concentration via ultrafiltration, and detailed steps for both protein and peptide fractionation kits.
| Kit pH range (Protein) | pH 4-7 |
|---|---|
| Kit pH range (Peptide) | pH 3-10 |
| Well setup configuration | 24-well setup |
| Incubation temperature (Digestion) | 37 °C |
Combining these approaches allows for a more in-depth characterization of plasma samples, identifying two- to threefold more proteins and three- to fourfold more peptides compared to controls without fractionation.
The method depletes six high abundant proteins: albumin, IgG, IgA, haptoglobulin, transferrin, and antitrypsin.