The Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells is an easy-to-use, comprehensive kit for preparation of clean peptide mixtures from cultured cells for mass spectrometry (MS) analysis.
The Pierce Mass Spec Sample Prep Kit for Cultured Cells contains all the necessary reagents and enzymes to prepare up to 20 samples (1 million cells each) for MS analysis. The simple and robust workflow uses a lysis protocol that generates approximately 100μg of protein per sample and includes a control digestion indicator to monitor the efficiency of the two-step enzymatic digestion protocol. The kit procedure, which includes a Digestion Indicator as an internal protein/peptide control, produces consistently and reproducibly clean peptide mixtures for protein identification.
Highlights:
- Complete – includes all reagents, a digestion indicator control, proteases and an optimized protocol needed to process up to 20 samples
- Simple – user friendly kit can provide reproducible results even for non-expert MS analysts
- Flexible – can be adapted to handle sample sizes between 10 to 200μg
- High yield – total protein yield from 1 million cells is greater than 100μg
- Optimized – cysteine reduction and alkylation are 100% with less than 1% over alkylation of non-cysteine residues
- Efficient - percentage of missed cleavages is less than 10%
- Compatible – final preparation is ready for direct MS analysis and other downstream applications, including mass-tag labeling
Includes:
Kit contains proprietary lysis and digestion buffers, digestion indicator, DTT, iodoacetamide, Lys-C, trypsin
Product Details:
Sample prep for mass spectrometry (MS, mass spec) remains one of the largest bottlenecks associated with MS analysis, and consistent and reproducible sample preparation can make the difference between a successful analysis and a failed outcome. Current sample prep protocols are primarily homebrew and can be highly variable, making data analysis and interpretation difficult. The Mass Spec Sample Prep Kit for Cultured Cells provides researchers with all the necessary tools to generate consistent and reproducible protein digests that are directly compatible with LC-MS workflows. The digests do not require further processing, such as C18 clean up or detergent removal.
The Pierce Digestion Indicator, which can be purchased separately, is a non-mammalian recombinant protein (26kDa) with five signature peptides for use in determining the digestion efficiency and reproducibility across multiple samples. The protein sequence and recommended peptides to monitor across samples are supplied with the product instructions.
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| Protocol summary for the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells. All reagent and enzymes are supplied in the kit, except trifluoroacetic acid (TFA), phosphate-buffered saline (PBS), and acetone. Pierce Universal Nuclease for Cell Lysis (Part No. 88700) can be used as an alternative to sonication for cell lysis. |
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| Comparison of protein yields by four sample prep methods. HeLa S3 cell pellets, each containing 2 x 10^6 cells, were resuspended (in duplicate) in 0.2mL of 0.1M Tris-HCl, 4% SDS, 0.1M DTT, pH 7.6 (FASP; Ref.1), 0.05M ammonium bicarbonate, 0.1% SDS, pH 8.0 (AMBIC-SDS; Ref.2), 0.1M Tris-HCl, 8M urea (Urea), pH 8.5 and the Lysis Buffer from the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Cultured Cells (Kit Buffer). Samples were incubated at 95°C for 5 minutes except the Urea sample, which was incubated at RT for 30 minutes. Each cell suspension was sonicated on ice for 20 seconds (pulse time 5 sec, pulse off time 5 sec, output level 2) using a Misonix* 3000 Sonicator. The cell debris was removed by centrifugation at 16,000 x g for 10 minutes and the supernatant was assayed for protein concentration using Pierce BCA Protein Assay (Part No. 23227) or Pierce BCA Protein Assay Kit-Reducing Agent Compatible (Part No. 23250). |
| Consistent, clean and reproducible LC-MS/MS results of three biological replicates. HeLa cell lysate (200μg) in 200μL lysis buffer and spiked with 1μg Digestion Indicator was processed by the Thermo Scientific Pierce Mass Spec Sample Prep Kit for Culture Cells and then analyzed by mass spectrometry. |
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Sample 1 |
Sample 2 |
Sample 3 |
| Number of Proteins |
3382 |
3228 |
3376 |
| Number of Unique Peptides |
16333 |
15939 |
17048 |
| Missed Cleavages (%) |
<10 |
<10 |
<10 |
| Disulfide Bond Reduction (%) |
100 |
100 |
100 |
| Cysteine Alkylation (%) |
100 |
100 |
100 |
| Over Akylation (%) |
0.1 |
0.3 |
0.9 |
| Digestion Indicator Protein Sequence Coverage (%) |
62.50 |
62.93 |
65.09 |
| Method Details: A Thermo Scientific EASY-nLC 1000 HPLC system and Thermo Scientific EASYSpray Source with Thermo Scientific EasySpray Column (25cm x 75μm i.d., PepMap, C18) was used to separate peptides (500ng) with a 30% acetonitrile gradient in 0.1% formic acid over 100min at a flow rate of 300nL/min. The samples were analyzed using Thermo Scientific Q Exactive hybrid quadrupole-Orbitrap mass spectrometer. For data analysis Thermo Scientific Proteome Discoverer software version 1.3 was used to search MS/MS spectra against the uniprot.human database using SEQUEST* search engine with a 1% false discovery rate. Static modifications included carbamidomethyl (C) and dynamic modifications included oxidation (M). The data set was screened by Preview* software from Protein Matrics* for sample preparation quality. To assess the digestion efficiency, the sequence of Digestion Indicator protein was included in the protein database. Five digestion indicator peptides were quantified manually with extracted ion-chromatograms of the raw LC-MS/MS data or automatically with Thermo Scientific Pinpoint software. |
References:
- Wisniewski, JR., et al. (2009). Universal sample preparation method for proteome analysis. Nat. Methods. 6:359-60.
- Bereman, MS., Egertson, JD., MacCoss, MJ. (2011). Comparison between procedures using sodium dodecyl sulfate for shotgun proteomic analyses of complex samples. Proteomics 11:2931-5.
Related Resources:
Mass Spectrometry Sample Preparation Handbook
Review of mass spectrometry
Related Products:
In-Solution Tryptic Digestion Kit
In-Gel Tryptic Digestion Kit
Pierce C-18 Spin Columns
DTT (dithiothreitol)
Proteases for Mass Spectrometry
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