Here, we establish a rapid, robust, and high reproducible workflow (see Fig. 8) to remove the more abundant proteins from serum
samples using only 30 μL of the sample. The workflow is very
interesting because they may be useful in clinical application, due
to the small sample amounts.
Our strategy removes most high-abundance plasma proteins
using a brief incubation with a reducing agent such as DTT added
directly to plasma at room temperature followed by removal of the
precipitate via centrifugation [61, 63].
1. Three aliquots of 30 μL of human serum are depleted.
2. To each sample, 3.33 μL of fresh 500 mM DTT is added,
mixed, and vortexed briefly.
3. The samples are incubated until a viscous white precipitate
persists (60 min).
4. The samples are centrifuged at 14,000 Â g for 20 min.
5. The supernatants are transferred to a clean tube.
6. Submit the sample to an in-gel concentration and in-gel
digestion.
3.2.2 Nanoparticles for
Protein Enrichment
Sample preparation is a fundamental step in most analytical procedures, particularly for the analysis of complex samples (biological
and environmental) [64, 65]. Many endogenous low-abundance
proteins/peptides suffer strong interference with highly abundant
Fig. 8 The serum proteome analysis using SWATH-MS. Serum samples were depleted using DTT reduction
before in-gel digestion
SWATH-MS Protocols in Human Diseases
121
samples using only 30 μL of the sample. The workflow is very
interesting because they may be useful in clinical application, due
to the small sample amounts.
Our strategy removes most high-abundance plasma proteins
using a brief incubation with a reducing agent such as DTT added
directly to plasma at room temperature followed by removal of the
precipitate via centrifugation [61, 63].
1. Three aliquots of 30 μL of human serum are depleted.
2. To each sample, 3.33 μL of fresh 500 mM DTT is added,
mixed, and vortexed briefly.
3. The samples are incubated until a viscous white precipitate
persists (60 min).
4. The samples are centrifuged at 14,000 Â g for 20 min.
5. The supernatants are transferred to a clean tube.
6. Submit the sample to an in-gel concentration and in-gel
digestion.
3.2.2 Nanoparticles for
Protein Enrichment
Sample preparation is a fundamental step in most analytical procedures, particularly for the analysis of complex samples (biological
and environmental) [64, 65]. Many endogenous low-abundance
proteins/peptides suffer strong interference with highly abundant
Fig. 8 The serum proteome analysis using SWATH-MS. Serum samples were depleted using DTT reduction
before in-gel digestion
SWATH-MS Protocols in Human Diseases
121