A single lysis solution for the analysis of tissue samples by different proteomic technologies

Abstract

Cancer, being a major healthcare concern worldwide, is one of the main targets for the application of emerging proteomic technologies and these tools promise to revolutionize the way cancer will be diagnosed and treated in the near future. Today, as a result of the unprecedented advances that have taken place in molecular biology, cell biology and genomics there is a pressing need to accelerate the translation of basic discoveries into clinical applications. This need, compounded by mounting evidence that cellular model systems are unable to fully recapitulate all biological aspects of human dissease, is driving scientists to increasingly use clinically relevant samples for biomarker and target discovery. Tissues are heterogeneous and as a result optimization of sample preparation is critical for generating accurate, representative, and highly reproducible quantitative data. Although a large number of protocols for preparation of tissue lysates has been published, so far no single recipe is able to provide a "one-size fits all" solubilization procedure that can be used to analyse the same lysate using different proteomics technologies. Here we present evidence showing that cell lysis buffer 1 (CLB1), a lysis solution commercialized by Zeptosens [a division of Bayer (Schweiz) AG], provides excellent sample solubilization and very high 2D PAGE protein resolution both when using carrier ampholytes and immobilized pH gradient strips. Moreover, this buffer can also be used for array-based proteomics (reverse-phase lysate arrays or direct antibody arrays), allowing the direct comparison of qualitative and quantitative data yielded by these technologies when applied to the same samples. The usefulness of the CLB1 solution for gel-based proteomics was further established by 2D PAGE analysis of a number of technically demanding specimens such as breast carcinoma core needle biopsies and problematic tissues such as brain cortex, cerebellum, skeletal muscle, kidney cortex and tongue. This solution when combined with a specific sample preparation technique - cryostat sectioning of frozen specimens - simplifies tissue sample preparation and solves most of the difficulties associated with the integration of data generated by different proteomic technologies.

Figure 1

Frozen tissue biopsy sample preparation workflow for gel‐based and array‐based proteomics.

Figure 2

Two‐dimensional gel electrophoresis protein patterns of whole protein extracts prepared from a needle core biopsy collected from a breast ductal carcinoma. Proteins were solubilized in O'Farrell lysis solution (A) or in CLB1 solution (B). Proteins were separated by 2D PAGE‐IEF and gels stained with silver nitrate. Several proteins that are differentially represented in the two samples are indicated by red arrows (lower expression in CLB1 lysis reagent) or blue arrows (higher expression in CLB1 lysis reagent). The identities of these proteins were ascertained by MALDI‐TOF and are presented in Table 1. The location of actin and albumin are also indicated for reference.

Figure 3

IEF 2D gel of whole tissue proteins extracted with O'Farrell lysis solution (left‐hand images) or CLB1 lysis solution (right‐hand images) from human brain cortex, cerebellum, kidney cortex, skeletal muscle and tongue. Solubilization of tissue specimens was carried out as described in Section 2.

Figure 4

IPG‐2D PAGE of whole protein extracts from a breast carcinoma sample run using IPG 4–7 (A) and 5–8 (B) strips. Proteins were solubilized in IPG lysis solution (BioRad) (left‐hand panel) or in CLB1 solution (right‐hand panel). The first dimension was carried out with IPG strips (BioRad) rehydrated in either IPG rehydration solution (BioRad) or in CLB1 solution (left‐ and right‐hand panels, respectively). The gels were stained with silver nitrate. The identity of a few proteins is indicated for reference.

Figure 5

Quantification of the levels of CK19 in three breast cancer samples using (A) 2D PAGE and (B) reverse‐phase lysate arrays. The bar graph plots (low right panels) show the ratio of the levels of CK19 normalized to the level in sample 1 (S1).

Figure 6

Compatibility of the CLB1 solution with the antibody protein microarray format. The tissue sample was frozen‐sectioned and solubilized in either Panorama® Antibody Microarray Extraction buffer or in CLB1 lysis solution and labeled with Cy3™ or Cy5™ dyes, respectively. The labeled samples were mixed and probed with the Panorama® signaling antibody array (Sigma‐Aldrich).