FAQs

For label-free quantitative analyses, we most often use in-solution digestion because this affords the least amount of sample-to-sample variability. We also recommend the analysis of in-solution digests for obtaining a qualitative "parts list" of the most abundant proteins in your sample. In-solution digestion and subsequent analysis has the advantage of avoiding bias due to extraction efficiency from the gel, and better raw sensitivity for samples where very little sample is available. We have performed in-solution digestion and analysis of samples with as little as 0.1 ug of protein.

The most common application of SDS-PAGE, in combination with LC-MS/MS (GeLC) is to qualitatively identify the composition of a particular gel band(s), following immunoprecipitation or protein purification. There are numerous other scenarios where gel-based analysis can be useful. For example, GeLC can be used to exclude one or more particularly high-abundance species from the analysis. This is useful in many co-immunoprecipitation studies where the bait protein is present at much higher levels than its interactors and would otherwise interfere with the detection of low-abundance interacting proteins

For gel bands, we prefer to receive them stored at 4C in either 50 mM AmBic or water, in a 1.5 mL eppendorf tube. For a solution submission, we prefer the sample already be in 50 mM Ammonium Bicarbonate. If this is not possible, please give us as many details as possible on the sample submission page. We need to know how to properly clean up the sample if it is in any other buffer, so it is critical that we know ALL OF THE BUFFER COMPONENTS. We have protocols available for the removal of many MS incompatible buffer components (some are less compatible than others), including the following:

• Buffers: PBS, Tris-HCl, HEPES, MES, MOPS, etc.
• Detergents: Triton X 100, NP40, SDS
• Other solvents: TCA, EtOH, MeOH, formamide, acetone

Although samples can be analyzed directly from an SDS-PAGE gel band if needed, it is most often preferred to work directly from solution as digestion efficiency is higher and peptides do not need to be extracted from the gel band. Note: phosphatase inhibitors such as KF and sodium orthovanadate are compatible with the enrichment-LC-MS protocol and should be included during sample preparation.

In nearly all circumstances, phosphorylated peptides exist in a much lower stoichiometry than non-phosphorylated peptides and, as such, require an enrichment procedure prior to LC-MS analysis. To perform our TiO2-based phosphopeptide enrichment protocol and be left with enough material for downstream LC-MS analysis, we ask that users provide a minimum of 10-15 ug total protein of a more pure (≥80% based on a coomassie standed SPD-PAGE) sample or 20-3 ug total protein of a less pure (≤50% based on coomassie stained SDS-PAGE) sample to maximize the chances of success. 

For an in-solution digestion and analysis, we ask for up to 10ug for LC-MS/MS if possible, based on a BSA-calibrated Bradford assay. We can many times complete an analysis with less; we aim to load 100ng to 1ug total protein onto our system for each run but require more due to sample losses during preparation. For gel bands, we have over 95% success rate for positive identification if the band is visible on a coomassie-stained gel. We have about a 75% success rate with silver-stained gels because of the higher sensitivity and sample losses that occur with this staining technique.

Keratin is quite common in protein analysis since it is very hard not to have some degree of skin shedding when preparing samples. Even if your sample is not human, we include common contaminants to your database, such as keratins so those proteins might appear in your analysis. To avoid keratins as much as possible, avoid handling your samples with bare hands, tie your hair back, avoid touching your skin with your gloves, do not wear wool, wear a lab coat. If at all possible, process your samples in a hood or consider your samples as "cell cultures" to avoid contaminations.