LC-MS Protein Sample Preparation Kits

EasyPep kits are standalone mass spec sample preparation kits that are manual or in some cases ready for automation processing in an open system format.

AccelerOme kits are fully automated and designed exclusively for use with the AccelerOme Automated Sample Preparation Platform.

Designed to simplify and standardize protein sample preparation, Thermo Scientific EasyPep Kits include unique and optimized reagents that condense the timeline to generate peptides suitable for mass spectrometry analysis.

Highlights:

• Comprehensive—includes pre-formulated reagents for lysis through digestion, peptide cleanup columns, and an optimized protocol that standardizes the process creating a reproducible sample preparation process from one experiment to another and from one lab to another
• Time-saving—sample processing has been reduced from more than 1 day to less than 4 hours
• Optimized—streamlined protocol and unique reagents minimize the number of steps and time to process samples
• Flexible formats—reagents and protocol have been verified using cells, plasma, serum, tissue, and purified protein samples from 1 µg to 2 mg

Time-savings

The number of steps and time have been reduced through multiple optimizations. First, Thermo Scientific Pierce Universal Nuclease replaces sonication to reduce viscosity from nucleic acids. Second, a collapsed and rapid incubation for cysteine modification has been employed with a “one pot” reduction/alkylation solution. Third, protein digestion has improved efficiency through the development of an optimized trypsin/Lys-C protease mixture to sample ratio. Finally, the kits include peptide cleanup columns (or plates) and buffers to prepare detergent-free peptide samples ready for mass spectrometry analysis in less than 4 hours.

Figure 1. Comparison of sample preparation for mass spectrometry analysis, using a traditional protein sample preparation workflow and EasyPep MS Sample Prep kits. Compared to a traditional workflow, the EasyPep MS Sample Prep kits provide the same high-quality data in less time and with half the number of manipulation steps. The optional step for TMTpro peptide labeling is indicated in both workflows.

Protocol and reagents

Compared to other methods and commercial kits, the EasyPep MS sample prep kits produce peptides consistent with mass spectrometry analysis, having higher protein yields and more unique protein group identifications. This is due to the high quality of the samples, which have fewer contaminants and higher digestion efficiency compared to traditional overnight digestion with trypsin alone.

Figure 2. HeLa cell lysates prepared using the EasyPep Mini MS Sample Prep Kit compared to other commercially available kits. The EasyPep Mini MS Sample Prep Kit provides similar reduction and alkylation efficiencies with more protein identifications and better digestion efficiency (higher percentage of zero missed cleavages) than other methods per microgram of sample analyzed. Thermo Scientific Pierce HeLa Protein Digest Standard (Cat. No. 88328) was used as a control sample.

Multiple formats

The EasyPep MS sample prep kits are available in five formats to support a wide range of sample numbers and input amounts.

The EasyPep Maxi MS Sample Prep Kit is optimized to process eight 0.5–2 mg protein samples excellent for phosphopeptide enrichment and other applications that require larger starting sample amounts. Alternatively, each Peptide Clean-Up Maxi Column can be used to process a combined set of isobaric tag-labeled samples (10–100 ug each, ≤2 mg total) for multiplex proteomic quantitation.

The EasyPep Mini and 96 MS Sample Prep kits are optimized to efficiently process protein samples of 10–100 µg with a high yield of mass spectrometry-ready peptides.

The EasyPep 96 Micro MS Sample Prep Kit can process 96 protein samples of 1–10 µg in parallel for increased throughput. Included in the kit is a peptide clean-up plate and solutions to prepare detergent-free peptide samples for LC-MS analysis or further sample processing such as isobaric tag (e.g., TMT reagent) labeling, and high pH reversed-phase fractionation with TMT/TMTpro pooled samples.

EasyPep Magnetic MS Sample Prep kits are developed for sample preparation processing using magnetic beads to bind and isolate proteins either on manual stands or on automated sample preparation platforms, including the KingFisher Instrument Platform.

Figure 3. The EasyPep MS sample prep kits use the same chemistry and standardized method to process purified proteins, cells, plasma, serum and tissues for downstream mass spectrometry-based proteomic applications. All formats can be used to generate samples for label-free quantitation, SureQuant targeted peptide assays, TMT reagent labeling, or phosphopeptide enrichment.

EasyPep Magnetic MS Sample Preparation kits

EasyPep Magnetic MS Sample Preparation kits are a mass spectrometry compatible magnetic bead sample preparation method. Our magnetic beads were specifically developed for mass spectrometry to be compatible with the harsh conditions of broad pH ranges and organic solvents. Other commercially available beads result in clumping and adherence to plastics used in the processing. Sample processing is the same as EasyPep column-based kits including lysis, nuclease, reduction, alkylation, digestion, binding and clean-up, but utilizing a magnetic bead procedure. A standard protocol, quality reagents, and specific beads designed for mass spectrometry protein sample preparation result in high reproducibility.

Magnetic bead sample preparation offers throughput options from manual processing to automation-friendly workflows including KingFisher instruments with protocols created for each instrument.

Automated sample preparation solution for mass spectrometry analysis

Break free from the long reaction times, high costs, and method complexity of manual proteomic sample preparation with the Thermo Scientific AccelerOme automated sample preparation platform. With factory-supplied reagents and kits and step-by-step onscreen instructions, high reproducibility and confident results are delivered for both label-free and tandem mass tag (TMT) multiplexing strategies with minimal effort providing several reagents to perform Label-free, TMT 11-plex, and TMT pro16-plex workflows at different sample scales. The AccelerOme platform is optimized to fit into the Thermo Scientific Orbitrap mass spectrometer ecosystem, streamlining the entire workflow from sample preparation to data processing.

Figure 5. Supporting kits and workflow of AccelerOme automated sample preparation system.