Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays

Abstract

Because most potential molecular markers and targets are proteins, proteomic profiling is expected to yield more direct answers to functional and pharmacological questions than does transcriptional profiling. To aid in such studies, we have developed a protocol for making reverse-phase protein lysate microarrays with larger numbers of spots than previously feasible. Our first application of these arrays was to profiling of the 60 human cancer cell lines (NCI-60) used by the National Cancer Institute to screen compounds for anticancer activity. Each glass slide microarray included 648 lysate spots representing the NCI-60 cell lines plus controls, each at 10 two-fold serial dilutions to provide a wide dynamic range. Mouse monoclonal antibodies and the catalyzed signal amplification system were used for immunoquantitation. The signal levels from the >30,000 data points for our first 52 antibodies were analyzed by using p-scan and a quantitative dose interpolation method. Clustered image maps revealed biologically interpretable patterns of protein expression. Among the principal early findings from these arrays were two promising pathological markers for distinguishing colon from ovarian adenocarcinomas. When we compared the patterns of protein expression with those we had obtained for the same genes at the mRNA level by using both cDNA and oligonucleotide arrays, a striking regularity appeared: cell-structure-related proteins almost invariably showed a high correlation between mRNA and protein levels across the NCI-60 cell lines, whereas non-cell-structure-related proteins showed poor correlation.

Fig. 1.

NCI-60 reverse-phase protein lysate microarrays. (A) Staining with SYPRO ruby for total protein. Each row (see enlarged image at the left) consists of 10 two-fold dilutions of an NCI-60 cell line or the control pool. The pool was spotted at four locations to control for pin effects. Concentrated pool was spotted at the bottom right corner of each field to serve as a registration mark for scanning. (B) CSA staining for p300 expression. (C) Negative control. (D) Representative candidate antibodies prescreened for specificity by Western blotting (20 μg per lane) with NCI-60 pool. *, bands at the predicted molecular weight. Blots 1–7 (from the left) show a single predominant band at the expected molecular weight. Blots 8–10 represent antibodies rejected for the array application because (i) the target band is fainter than other bands (lane 8); (ii) the target band is approximately equal in intensity to other bands (lane 9); and (iii) the target band is dominant (lane 10), but the other bands persist when the lysate is diluted to a point that the target band is below saturation (data not shown).

Fig. 2.

Analysis of p300 expression. (A) An array incubated with p300 primary antibody and stained by CSA. (B) Sixty four dilution curves in eight fields on the array. y axes, p-scan intensity of p300 signal; x axes, log₂(dilution factor). Numbers after cell line names are DI₂₅ values. The order of cell line listing corresponds to placement on the array. (C) DI₂₅ algorithm calculations for field 7. Broken line, the 25% level (at 43 units).

Fig. 3.

Clustered image map relating the expression levels of 52 proteins in the NCI-60 cell lines. Data were generated by using the DI₂₅ algorithm and data mean centered across both cells and proteins. (Right Lower Inset) The difference in c-erbB2 level between MDA-MB435 cells (DI₂₅ = –1.4) and MDA-N (DI₂₅ = +0.5). KRT, cytokeratins.

Fig. 4.

Transcript/protein correlation coefficients. The data were from two independent mRNA profiling platforms (cDNA and oligonucleotide arrays). Each point represents a single target protein. Molecular species were divided into two major categories according to SWISS-PROT and/or MIPS. Blue squares, cell-structure-related proteins; red squares, non-cell-structure-related proteins.