3. The sample is centrifuged at 18,840 Â g for 20 min.
4. The supernatants are transferred to a clean tube.
5. The sample is dried under vacuum for 15 min at 35
C in a
SpeedVac™ concentrator.
6. The sample is reconstituted in 30 μL of 25 mM Ambic.
NP Protein Alkylation and Fractionation
7. After protein depletion, the reduced SH-groups are alkylated
with 3 μL of 660 mM IAA (final IAA concentration 50 mM)
for 45 min at room temperature and protected from light.
8. After protein reduction and alkylation, 75 μL of AuNPs/
AgNPs/PtNPs is added to each serum aliquot.
9. Then, add 40 μL of citrate/citric acid buffer to a final pH
of 5.8.
10. NP-serum solution is incubated at 37
C with shaking
(600 rpm) in a thermostatic bath for 30 min.
11. The pellet is harvested by centrifugation at 18,840 Â g (AuNPs
and AgNPs) and 24,610 Â g (PtNPs) for 30 min.
12. The pellet is washed three times, with 25 μL of citrate/citric
acid buffer.
13. The pellet is harvested again by centrifugation at 18,840 Â g
(AuNPs and AgNPs) and 24,610 Â g (PtNPs) for 30 min to
remove unbound proteins.
Fe Nanoparticles
The synthesis of magnetic nanoparticles (FeNPs) is performed
according to a previously described procedure [114].
Depletion of Multiple High-Abundance Proteins
1. Fresh 500 mM DTT (3.3 μL) is mixed with 30 μL of human
serum and vortex briefly.
2. The sample is incubated until a viscous white precipitate persists (60 min).
3. The sample is centrifuged at 18,840 Â g for 20 min.
4. The supernatants are transferred to a clean tube.
5. The sample is dried under vacuum for 15 min at 35
C.
6. The sample is reconstituted in 30 μL of 25 mM Ambic.
NP Protein Alkylation and Fractionation
7. After protein depletion, the reduced SH-groups are alkylated
with 3 μL of 660 mM IAA for 45 min at room temperature and
protected from light.
8. After protein reduction and alkylation, 13 μL of serum aliquot
is transferred to a clean tube.
9. 5 μL of FeNPs is added to serum aliquot.
SWATH-MS Protocols in Human Diseases
125
4. The supernatants are transferred to a clean tube.
5. The sample is dried under vacuum for 15 min at 35
C in a
SpeedVac™ concentrator.
6. The sample is reconstituted in 30 μL of 25 mM Ambic.
NP Protein Alkylation and Fractionation
7. After protein depletion, the reduced SH-groups are alkylated
with 3 μL of 660 mM IAA (final IAA concentration 50 mM)
for 45 min at room temperature and protected from light.
8. After protein reduction and alkylation, 75 μL of AuNPs/
AgNPs/PtNPs is added to each serum aliquot.
9. Then, add 40 μL of citrate/citric acid buffer to a final pH
of 5.8.
10. NP-serum solution is incubated at 37
C with shaking
(600 rpm) in a thermostatic bath for 30 min.
11. The pellet is harvested by centrifugation at 18,840 Â g (AuNPs
and AgNPs) and 24,610 Â g (PtNPs) for 30 min.
12. The pellet is washed three times, with 25 μL of citrate/citric
acid buffer.
13. The pellet is harvested again by centrifugation at 18,840 Â g
(AuNPs and AgNPs) and 24,610 Â g (PtNPs) for 30 min to
remove unbound proteins.
Fe Nanoparticles
The synthesis of magnetic nanoparticles (FeNPs) is performed
according to a previously described procedure [114].
Depletion of Multiple High-Abundance Proteins
1. Fresh 500 mM DTT (3.3 μL) is mixed with 30 μL of human
serum and vortex briefly.
2. The sample is incubated until a viscous white precipitate persists (60 min).
3. The sample is centrifuged at 18,840 Â g for 20 min.
4. The supernatants are transferred to a clean tube.
5. The sample is dried under vacuum for 15 min at 35
C.
6. The sample is reconstituted in 30 μL of 25 mM Ambic.
NP Protein Alkylation and Fractionation
7. After protein depletion, the reduced SH-groups are alkylated
with 3 μL of 660 mM IAA for 45 min at room temperature and
protected from light.
8. After protein reduction and alkylation, 13 μL of serum aliquot
is transferred to a clean tube.
9. 5 μL of FeNPs is added to serum aliquot.
SWATH-MS Protocols in Human Diseases
125