EasyPep™ 96 MS Sample Prep Kit, 96 Reactions - FAQs

As per protocol, peptide samples need to be dried down prior to LC-MS analysis. This step can be performed using vacuum pumps/dry-down devices without any harm caused to the device by the components present in the elution buffer within the EasyPep MS Sample Prep Kits.

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The bulk Top14 depletion resin is compatible with a 96-well filter plate such as the Agilent filter microplate (Cat. No. 200957-100). Add 600 µL of resin slurry (50%) to each of the wells in the 96-well filter plate, add 10-20 µL of sample, and incubate with gentle shaking. Depleted samples can be collected in a new 96-well polypropylene plate by centrifugation for 2 mins at 1,000 x g or by using a vacuum manifold.

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High sample viscosity after lysis is due to release of DNA from the nucleus. Sonication or addition of a nuclease such as the Pierce Universal Nuclease (Cat. No. 88700, 88701, or 88702) can be used to degrade DNA and reduce sample viscosity.

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Add 0.5 mm diameter glass beads to the sample pelleted in Easy Pep Lysis Solution and proceed as recommended in the Easy Pep sample prep protocol. Vortex vigorously for few minutes, spin at 16,000 x g for 5 mins, and extract the supernatant for protein assay and further downstream sample preparation.

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Add 1-2 µL of 50 mg/mL Lysozyme Solution (Cat. No. 90082) to 200 µL of the EasyPep lysis buffer provided in the kit and proceed with the sample prep. Add 200 µL of the prepared lysis solution to 5 X 10E8 bacterial cells (E. coli cells) (OD600 equal to 1, corresponding to 1 X 10E8 CFUs). Lyse by pipetting the sample repeatedly and centrifuge at 16,000 x g for 5 mins. Alternatively, E. coli cells can be lysed in lysis buffer using bead beating or sonication instead of lysozyme. After lysis, dilute samples to 1 mg/mL using lysis buffer, proceed with the EasyPep protocol for reduction, alkylation, digestion, and clean up as described in the product manual.

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High Select Top14 Abundant Protein Depletion Mini Spin columns (Cat. Nos. A36369, A36370) can be used to deplete top 14 abundant plasma/serum proteins. The depletion of highly abundant proteins from the sample enables the detection and identification of low abundant proteins. The depleted samples can be processed with EasyPep MS sample prep kit to generate digested plasma/serum samples. The clean digested samples can be processed by nanoLC-MS/MS analysis for discovery experiments or nanoLC-PRM/MS analysis for targeted experiments.

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TMT labeling can be performed after reduction, alkylation, digestion, or prior to cleanup, if the sample is buffered at appropriate pH (8-8.5) in a suitable buffer without primary amines (e.g., Tris, glycine). TMT labeling can also be performed after peptide clean-up. Labeling of peptides after clean-up enables measuring and normalizing of the peptide samples for equal mixing.

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Peptide yield can be measured using the Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275) or the Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290). The choice of peptide assay depends on the sample type and composition of the sample buffer. The fluorometric peptide assay cannot be used to measure peptides with chemically modified amines such as acetylated peptides or TMT-labeled protein digests. The colorimetric assay can measure a wider range of samples but is not as sensitive as the fluorometric assay, requiring more sample for accurate detection. Finally, both assays are susceptible to interfering compounds in the sample or buffer which should be avoided or removed for best results.

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We don't recommend adding protease inhibitors as they can affect trypsin and trypsin/Lys-C activity. If protease inhibitors are present in the protein sample or cells, they should be removed by dialysis, diafiltration, desalting, or protein precipitation prior to enzymatic digestion. For phosphopeptide enrichment workflows, we recommend adding phosphatase inhibitors to the lysis solution prior to cell lysis.

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Applying correction factors is required for more accurate relative protein quantitation as the isotopic impurity is variable among the tags, ranging from 5-10%.

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This is most often done with the utilization of a "pool channel" for each multiplex set. This reference channel typically contains an equimolar mixture of all samples which is labeled with one of the TMT tags that is shared among the sets. The pool channel can be used to then normalize relative protein abundance measurements across multiplexed sets.

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The EasyPep kit chemistry is fully compatible with TMT/TMTpro reagents. Most commonly, TMT/TMTpro reagents are used to label peptides immediately after digestion and before peptide cleanup. This enables combining samples for a single cleanup step which reduces variability for more reproducible quantitative measurements. Another option is to label samples after peptide cleanup. This approach provides efficient labeling of the samples but may require the use of a peptide quantification assay to ensure that samples are equally mixed before LC-MS analysis. Both the workflows should provide high labelling efficiencies using our recommended TMT/TMTpro reagent to sample ratios.

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We recommend reviewing your sample-prep workflow to ensure consistent protein extraction, reduction/alkylation, digestion, and clean-up. We recommend using EasyPep products for high quality, reproducible sample preparation (EasyPep Maxi Sample Prep Kit, EasyPep Mini MS Sample Prep Kit, EasyPep 96 MS Sample Prep Kit). We also recommend quantifying peptides using the Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290) or Pierce Quantitative Coloimetric Peptide Assay (Cat. No. 23275) to ensure that the same amount of peptides are being used for each LC-MS analysis. Poor reproducibility could also be related to the LC-MS system performance which may require recalibration using Pierce Calibration Solutions. System performance can be assessed using protein digest standards such as Pierce HeLa Protein Digest Standard or Pierce TMT11plex Yeast Digest Standard and peptide standards such as Pierce Peptide Retention Time Calibration Mixture or Pierce LC-MS/MS System Suitability Standard (7 x 5 Mix).

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We offer EasyPep MS sample preparation kits (EasyPep Maxi Sample Prep Kit (Cat. No. A45734), EasyPep Mini MS Sample Prep Kit (Cat. No. A40006), EasyPep 96 MS Sample Prep Kit (Cat. No. A45733)) that contain wash buffers specifically formulated to clean up TMT-labeled protein digests. To remove excess TMT reagent from samples prepared using other sample preparation methods, we recommend using Pierce Peptide Desalting Spin Columns (Cat. Nos. 89851, 89852) with extra washes using 5% methanol. The Pierce High pH Reversed-Phase Peptide Fractionation Kit (Cat. No. 84868) can also be used to remove excess unreacted TMT tags before collecting fractions.

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The Sample Prep Plate and Wash and Elution Plate do not have lids. They are the standard polypropylene deep-well plates so any 96-well plate cover would work, or they can be covered with parafilm.

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First of all, we do not recommend re-using wells from the Peptide Clean-Up Plate that have already been used for clean-up. The remaining unused wells in the Peptide Clean-Up Plate can be used later as the wells contain a dry resin that will not be affected by vacuum or centrifugation steps. We recommend storing the plate at 4 degrees C, with unused wells sealed with parafilm or plate sealer film.

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The peptide clean-up columns will remove any undigested proteins which may remain in the sample including the enzymes provided in the EasyPep kit. A few peptides from the enzymes would end up in the final digest, but they do not typically interfere with proteomic analysis as the amount of enzymes compared to the sample amount is very low. When identifying peak IDs, these peptides will be identified by the database used in the analysis.

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For long-term storage (>3 months), we recommend storing the Trypsin/Lys-C Protease Mix at -20 degrees C. After addition of Enzyme Reconstitution Solution, the Trypsin/Lys-C Protease Mix can be stored at 4 degrees C for up to 1 month or at -20 degrees C for 1 year.

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Yes, serum and plasma are compatible with Easy Pep kits. We recommend diluting samples directly in Lysis Solution to a final total protein concentration in the range of 0.1-1 mg/mL.

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The digestion efficiency with the EasyPep Mini MS Sample Prep Kit is >90% with a high efficiency in alkylation/reduction.

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EasyPep MS sample prep kits can be used to process samples after immunoprecipitation. However, the EasyPep Lysis Buffer should not be used for immunoprecipitation as it contains a denaturing detergent that interferes with antigen binding.

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The EasyPep lysis buffer has been optimized to work with sequential chemistries, digestion, and clean up used in the EasyPep MS sample prep kits. We do not recommend using other lysis buffers as they may contain buffers or detergents that may interfere with reduction, alkylation, digestion, or peptide clean-up.

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The EasyPep Mini MS Sample Prep kit is compatible with TMT labeling, peptide fractionation, and phosphopeptide enrichment.

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EasyPep MS sample prep kits have been tested with mammalian cell lines (HeLa, A549, HEK293, CHO), fresh/frozen tissues (brain, heart, liver), human plasma and serum, bacteria (E.coli), yeast, and FFPE sections.
Note: FFPE sections need to be de-paraffinized and air dried before using the kit.

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