What Does the FFPE-FASP Protein Digestion Kit Do?
The FFPE-FASP Protein Digestion Kit:
• Reverses protein crosslinking that originates from formalinfixation
• Extracts the proteins from the tissue
• Reduces the disulfide bonds
• Depletes contaminants from any protein sample solution
• Alkylates thiols
• Digests proteins in a spin-filter “proteomic reactor”
• Releases peptide products of digestion in a small volume filtrate
• Excludes remaining large molecules such as DNA from the digestion
When Should I Use the FFPE-FASP Protein Digestion Kit?
Use this kit to analyze formalin-fixed tissue specimens for proteomics.
This kit provides the reagents necessary to reverse protein crosslinking,
extract the protein efficiently, and carry out digestion in a convenient
Is the FFPE-FASP Protein Digestion Kit Similar to the “Universal Sample Preparation” Method?
The FASP Protein Digestion Kit enables researchers to successfully reproduce the variant of the Universal Sample Preparation method
described by Ostasiewicz, et al. J Proteome Res. 2010 Jul 2;9(7):3688-700.
Proteome, phosphoproteome, and N-glycoproteome are quantitatively preserved in formalin-fixed paraffin-embedded tissue and analyzable by high-resolution mass spectrometry. Ostasiewicz P, Zielinska DF, Mann M, Wiśniewski JR.
Tissue samples in biobanks are typically formalin-fixed and paraffin-embedded (FFPE), in which form they are preserved for decades. It has only recently been shown that proteins in FFPE tissues can be identified by mass spectrometry-based proteomics but analysis of posttranslational
modifications is thought to be difficult or impossible. The filter-aided sample preparation (FASP) method can analyze proteomic samples solubilized in high concentrations of SDS and we use this feature to develop a simple protocol for FFPE analysis. Combination with simple pipet-tip based peptide fractionation identified about 5000 mouse liver proteins in 24 h measurement time-the same as in fresh tissue. Results from the FFPE-FASP procedure do not indicate any discernible changes due to storage time, hematoxylin staining or laser capture microdissection. We compared fresh against FFPE tissue using the SILAC mouse and found no significant qualitative or quantitative differences between these samples either at the protein or the peptide level. Application of our FFPE-FASP protocol to phosphorylation and Nglycosylation pinpointed nearly 5000 phosphosites and 1500 Nglycosylation sites. Analysis of FFPE tissue of the SILAC mouse revealed that these post-translational modifications were quantitatively preserved. Thus, FFPE biobank material can be analyzed by quantitative proteomics at the level of proteins and post-translational modifications.
What Additional Materials Do I Need?
In addition to the FFPE-FASP Protein Digestion Kit, you’ll need absolute ethanol, xylene (2mL each per tissue sample), 30 μg trypsin for each digestion and some common laboratory supplies and equipment. The package insert has a complete list for you to check before you start.
How Many Digests Can I Perform with One Kit?
The FFPE-FASP Protein Digestion Kit contains sufficient materials for
eight digests of up to 1.0 mg FFPE tissue.
What is the Shelf Life of the Kit?
The product shelf life is 6 months when stored at 4°C.
What is the expected yield obtained by the FASP-FFPE method?
The typical yields obtained by FASP-FFPE digestion are about 50% of the applied protein sample. This is true over a wide range of protein amounts from few micrograms (processed in 0.5 ml units) up to 20 milligrams (15 ml units). It has been reported that FASP is unique in allowing precise monitoring of the digest yield, since neither the in-gel nor the in-solution (urea/thiourea) methods enable analysis of the efficiency of digestion.
Why do we use 30,000 Da cut-off filters?
Denatured proteins remain unfolded in urea. Due to their large Stokes’ radii, they are retained in the concentrators by the filter during the buffer exchange procedure. Importantly, peptides with sizes up to 5,000 Da can be collected using 30,000 Da filters. In contrast, the units with cutoffs of 3,000 Da or 10,000 Da retain larger peptides. In addition, the time required for concentration using the 3,000 Da and 10,000 Da filters is several folds longer when compared to 30,000 Da filters. For these reasons, the 30,000 Da filters re superior. For reference see:Anal. Biochem. 410, 307 (2011).
What does it mean when the buffer is exchanged too slow or too fast?
Typically 10 – 15 minutes at 14,000 × g are required for concentration of the 0.2 ml 8 M urea solution into about 10 μl in a 0.5 ml filter unit. Applying too much protein lysate can result in clogging of the filters. This leads to increase of the required centrifugation times. If the urea solution passes through the filter too quickly (a minute or less), this is typically a sign of a membrane perforation.
Are there other proteases, other than trypsin, that are compatible with
The FASP-FFPE is compatible with all commonly used proteases. The original paper from Mann describes the use of FASP with Lys-C. PDI customers have also reported the use of our kits with pepsin. When changing to a protease that is different than trypsin, it is important to adjust the buffer conditions, enzyme/protein ratio, and reaction time to accommodate the change.
The protocol includes an optional elution step using NaCl. What is the purpose of this step?
The NaCl serves to improve recovery of the digested peptides during the final centrifugation step. The salt breaks any interaction between the remaining peptides and the spin filter membrane. This is optional and may be unnecessary if the protein is abundant or salt is problematic downstream.
Is this protocol compatible with Laser Microdissection?
Yes. This application has been demonstrated and published in the following journal article: J Proteome Res. 2011 Apr 28. [Epub ahead of print] High Recovery FASP Applied to the Proteomic Analysis of Microdissected ormalin Fixed Paraffin Embedded Cancer Tissues Retrieves Known Colon Cancer Markers. Wisniewski JR, Ostasiewicz P, Mann M.
Proteomic analysis of samples isolated by laser capture microdissection from clinical specimens requires sample preparation and fractionation methods suitable for small amounts of protein. Here we describe a streamlined filter-aided sample preparation (FASP) workflow that allows efficient analysis of lysates from low numbers of cells. Addition of carrier substances such as polyethylene glycol or dextran to the processed samples improves the peptide yields in the low to submicrogram range. In a single LC-MS/MS run, analyses of 500, 1000 and 3000 cells allowed identification of 905, 1536 and 2055 proteins, respectively. Incorporation of an additional SAX fractionation step at somewhat higher amounts enabled the analysis of formalin fixed and paraffin embedded human tissues prepared by LCM to a depth of 3,600-4,400 proteins per single experiment. We applied this workflow to compare archival neoplastic and matched normal colonic mucosa cancer specimens for three patients. Label-free quantification of more than 6,000 proteins verified this technology through the differential expression of 30 known colon cancer markers. These included Carcino-Embryonic Antigen (CEA), the most widely used colon cancer marker, complement decay accelerating factor (DAF, CD55) and Metastasis-associated in colon cancer protein 1 (MACC1). Concordant with literature knowledge, mucin 1 was overexpressed and mucin 2 underexpressed in all three patients. These results show that FASP is suitable for the low level analysis of microdissected tissue and it has the potential for exploration of clinical samples for biomarker and drug target discovery.