5. Pellet agarose beads by centrifugation for 4 min at 300 Â g,
remove supernatant, and wash beads with 1.8 mL deionized
water.
6. Resuspend beads in 1 mL SDS wash buffer and reduce beadbound proteins with 5 mM TCEP for 15 min at 55
C and
15 min at room temperature.
7. Pellet beads at 300 Â g for 4 min to remove supernatant.
8. Alkylate the proteins with 40 mM iodoacetamide for 30 min, at
room temperature, in the dark.
9. Transfer beads to Mobicol classic 35-μm filters and wash beads
with 10 mL of the following buffers: SDS wash buffer, urea
wash buffer, 5 M NaCl, 80% isopropanol, 100 mM NaHCO 3 ,
50 mM ammonium bicarbonate (60
C), and 20% acetonitrile.
10. Transfer the beads in fresh Mobicol tube.
3.4.4 On-Bead Trypsin
Digestion
1. Resuspend the beads in 400 μL digestion buffer.
2. Add 1 μg sequencing grade modified trypsin and incubate for
16 h at 37
C.
3. Collect the peptides, wash beads twice with 50 mM ammonium bicarbonate.
4. Acidify samples with 10% formic acid to pH 3.
3.4.5 Peptide Purification
1. Desalt the peptides using UltraMicroSpin C18 Columns with
5–60 μg capacity for tryptic peptide fraction according to
manufacturer’s instructions.
2. Dry the eluted peptides in a SpeedVac and store them at
À80
C until further analysis.
3.4.6 LC-MS/MS and
Data Analysis
LRC samples are of medium-to-high complexity and need to be
analyzed with a highly sensitive, high mass accuracy mass spectrometer. Analyze peptides with a standard shotgun mass spectrometrybased workflow and perform label-free quantification to extract
relative protein abundance.
3.4.7 Statistical Analysis
Perform statistical analysis to calculate protein fold changes and
their statistical significance between paired conditions; numerous
free software packages exist for statistical analysis such as MSstats
[12] and SafeQuant [13].
3.4.8 Data Visualization
and Interpretation
A volcano plot combines a measure of statistical significance from a
statistical test with the magnitude of the change, enabling quick
visual identification of proteins that were significantly enriched in
the ligand of interest samples. The x-axis represents the mean ratio
fold change (on a log2 scale). The y-axis represents the statistical
significance p-value of the ratio fold change for each protein (on a
Validating Cardiac Targeting Peptide
109
remove supernatant, and wash beads with 1.8 mL deionized
water.
6. Resuspend beads in 1 mL SDS wash buffer and reduce beadbound proteins with 5 mM TCEP for 15 min at 55
C and
15 min at room temperature.
7. Pellet beads at 300 Â g for 4 min to remove supernatant.
8. Alkylate the proteins with 40 mM iodoacetamide for 30 min, at
room temperature, in the dark.
9. Transfer beads to Mobicol classic 35-μm filters and wash beads
with 10 mL of the following buffers: SDS wash buffer, urea
wash buffer, 5 M NaCl, 80% isopropanol, 100 mM NaHCO 3 ,
50 mM ammonium bicarbonate (60
C), and 20% acetonitrile.
10. Transfer the beads in fresh Mobicol tube.
3.4.4 On-Bead Trypsin
Digestion
1. Resuspend the beads in 400 μL digestion buffer.
2. Add 1 μg sequencing grade modified trypsin and incubate for
16 h at 37
C.
3. Collect the peptides, wash beads twice with 50 mM ammonium bicarbonate.
4. Acidify samples with 10% formic acid to pH 3.
3.4.5 Peptide Purification
1. Desalt the peptides using UltraMicroSpin C18 Columns with
5–60 μg capacity for tryptic peptide fraction according to
manufacturer’s instructions.
2. Dry the eluted peptides in a SpeedVac and store them at
À80
C until further analysis.
3.4.6 LC-MS/MS and
Data Analysis
LRC samples are of medium-to-high complexity and need to be
analyzed with a highly sensitive, high mass accuracy mass spectrometer. Analyze peptides with a standard shotgun mass spectrometrybased workflow and perform label-free quantification to extract
relative protein abundance.
3.4.7 Statistical Analysis
Perform statistical analysis to calculate protein fold changes and
their statistical significance between paired conditions; numerous
free software packages exist for statistical analysis such as MSstats
[12] and SafeQuant [13].
3.4.8 Data Visualization
and Interpretation
A volcano plot combines a measure of statistical significance from a
statistical test with the magnitude of the change, enabling quick
visual identification of proteins that were significantly enriched in
the ligand of interest samples. The x-axis represents the mean ratio
fold change (on a log2 scale). The y-axis represents the statistical
significance p-value of the ratio fold change for each protein (on a
Validating Cardiac Targeting Peptide
109