Table 2.
Summary of Common Problems Encountered and Their Solutions
| Problem | Solution |
|---|---|
| Basic Protocol | |
| Sample clogged on S‐Trap™ | Centrifuge the sample thoroughly before pipetting, and be sure to shear DNA with sonication or with the addition of MgCl2 and Benzonase. |
| Contaminants in MS data, such as polyethylene glycol or lipids, or high backpressure during sample loading | Perform additional wash steps on S‐trap™ at step 14. For removal of lipids, add three washes of 50:50 (v/v) methanol/chloroform before standard S‐trap™ buffer washes. |
| Percentage of peptides identified with at least one missed cleavage is >15% | Increase the concentration of trypsin if digesting a large amount of protein. |
| Alternate Protocol 1 | |
| Sample contaminated with detergents or polymers, as evidenced by poor LCMS data | Use detergent‐free glassware; be sure to perform SP2 cleanup, as C18 cleanup protocols do not remove PEG. |
| Large number of missed cleavages in database search results | Check that the digestion solution pH is ∼8 after adding trypsin to the protein, and adjust with small amounts of concentrated acid or base as necessary. |
| Alternate Protocol 2 | |
| Low peptide recovery |
During particle preparation, let particles warm to room temperature under constant shaking on an orbital shaker for ≥30 min. Sufficiently suspend particles by shaking and vortexing to yield a homogenous suspension. Particle preparation should be made fresh every 6 months. Ensure that math was performed correctly during peptide quantification assay. Alternatively, repeat peptide quantification assay. To maximize recovery, use 0.2‐0.4 µl of 50 µg/µl particle suspension per microgram of peptide to be cleaned (i.e., a particle to peptide ratio of 10:1‐20:1). |
| PEG or other contaminants in MS data |
Use detergent‐free glassware. Make fresh elution solution. |
| Support Protocols 1 and 2 | |
| No protein in sample, or low protein concentration after lysis | Use ionic detergent compatibility reagent included with the Pierce kit. |
| Variable numbers in duplicate values | Be sure to allow the reagents to come to room temperature. Consider checking pipet technique. |
| No protein in sample, or low protein concentration after tissue lysis | Be sure to use ionic detergent compatibility reagent included in the Pierce kit if there are detergents in the sample. |
| Too much protein for standard range | Dilute sample further with lysis buffer before attempting protein quantification again. |
| Support Protocol 3 | |
| Variable peptide amounts in fractions | Consider changing acetonitrile percentages to optimize spread of peptides. |
| No peptides in any fractions | Check the saved flowthrough and wash tubes for non‐retained peptides. |