4. Add 8 μL of 5% hydroxylamine to the sample and incubate for
15 min to quench the reaction.
5. After quenching the labeling reaction, combine samples in a
new tube at equal amounts.
6. Vortex and spin the tube.
7. Calculate the total volume of the sample and divide into new
tubes by adding to each 100 μg of protein digest (see Note 26).
8. Dry the aliquots in a vacuum centrifuge (see Note 27).
9. Use one aliquot to quantitative colorimetric peptide assay to
measure protein digest concentration (see Note 28).
3.6 Peptide Cleanup
and Fractionation
1. Fractionate two aliquots in case of need to repeat LC–MS/MS
analysis.
2. Prepare the elution solutions for TMT-labeled peptides according to Table 1.
3.6.1 Conditioning
of the Spin Columns
1. Remove the protective white tip from the bottom of the column and discard. Place the column into a 2 mL sample tube.
2. Centrifuge at 5000 Â g for 2 min to remove the solution and
pack the resin material. Discard the liquid (see Note 29).
3. Remove the top screw cap and load 300 μL of ACN into the
column. Replace the cap, place the spin column back into a
2 mL sample tube, and centrifuge at 5000 Â g for 2 min.
Discard ACN and repeat wash step.
4. Wash the spin column twice with 0.1% TFA solution, as
described in step 3. The column is now conditioned and
ready for use.
3.6.2 Fractionation
of Peptide Samples
1. Dissolve 100 μg of the digested sample (Subheading 3.4, step
8) in 300 μL of 0.1% TFA solution.
2. Place the spin column into a new 2.0 mL sample tube. Load
300 μL of the sample solution onto the column, replace the top
cap, and centrifuge at 3000 Â g for 2 min. Retain eluate as a
“flow-through” fraction (see Note 30).
3. Place the column into a new 2 mL sample tube. Load 300 μL of
water onto the column and centrifuge again to collect the wash.
Retain eluate as a “wash water” fraction.
4. Place the column into a new 2 mL sample tube. Load 300 μL of
5% ACN, 0.1% TEA (see Table 1) to remove unreacted TMT
reagent. Retain eluate as a “wash ACN” fraction.
5. Place the column into a new 2.0 mL sample tube. Load 300 μL
of the appropriate elution solution (e.g., 10% ACN, 0.1% TEA)
and centrifuge at 3000 Â g for 2 min to collect the fraction.
Retain eluate as a “1” fraction.
Shotgun Proteomics for Anisakis simplex
69
15 min to quench the reaction.
5. After quenching the labeling reaction, combine samples in a
new tube at equal amounts.
6. Vortex and spin the tube.
7. Calculate the total volume of the sample and divide into new
tubes by adding to each 100 μg of protein digest (see Note 26).
8. Dry the aliquots in a vacuum centrifuge (see Note 27).
9. Use one aliquot to quantitative colorimetric peptide assay to
measure protein digest concentration (see Note 28).
3.6 Peptide Cleanup
and Fractionation
1. Fractionate two aliquots in case of need to repeat LC–MS/MS
analysis.
2. Prepare the elution solutions for TMT-labeled peptides according to Table 1.
3.6.1 Conditioning
of the Spin Columns
1. Remove the protective white tip from the bottom of the column and discard. Place the column into a 2 mL sample tube.
2. Centrifuge at 5000 Â g for 2 min to remove the solution and
pack the resin material. Discard the liquid (see Note 29).
3. Remove the top screw cap and load 300 μL of ACN into the
column. Replace the cap, place the spin column back into a
2 mL sample tube, and centrifuge at 5000 Â g for 2 min.
Discard ACN and repeat wash step.
4. Wash the spin column twice with 0.1% TFA solution, as
described in step 3. The column is now conditioned and
ready for use.
3.6.2 Fractionation
of Peptide Samples
1. Dissolve 100 μg of the digested sample (Subheading 3.4, step
8) in 300 μL of 0.1% TFA solution.
2. Place the spin column into a new 2.0 mL sample tube. Load
300 μL of the sample solution onto the column, replace the top
cap, and centrifuge at 3000 Â g for 2 min. Retain eluate as a
“flow-through” fraction (see Note 30).
3. Place the column into a new 2 mL sample tube. Load 300 μL of
water onto the column and centrifuge again to collect the wash.
Retain eluate as a “wash water” fraction.
4. Place the column into a new 2 mL sample tube. Load 300 μL of
5% ACN, 0.1% TEA (see Table 1) to remove unreacted TMT
reagent. Retain eluate as a “wash ACN” fraction.
5. Place the column into a new 2.0 mL sample tube. Load 300 μL
of the appropriate elution solution (e.g., 10% ACN, 0.1% TEA)
and centrifuge at 3000 Â g for 2 min to collect the fraction.
Retain eluate as a “1” fraction.
Shotgun Proteomics for Anisakis simplex
69