A novel phosphoprotein analysis scheme for assessing changes in premalignant and malignant breast cell lines using 2D liquid separations, protein microarrays and tandem mass spectrometry

Abstract

An analysis of phosphorylation changes that occur during cancer progression would provide insights into the molecular pathways responsible for a malignant phenotype. In this study we employed a novel coupling of 2D-liquid separations and protein microarray technology to reveal changes in phosphoprotein status between premalignant (AT1) and malignant (CA1a) cell lines derived from the human MCF10A breast cell lines. Intact proteins were first separated according to their isoelectric point and hydrophobicities, then arrayed on SuperAmine glass slides. Phosphoproteins were detected using the universal, inorganic phospho-sensor dye, ProQ Diamond. Using this dye, out of 140 spots that were positive for phosphorylation, a total of 85 differentially expressed spots were detected over a pH range of 7.2 to 4.0. Proteins were identified and their peptides sequenced by mass spectrometry. The strategy enabled the identification of 75 differentially expressed phosphoproteins, from which 51 phosphorylation sites in 27 unique proteins were confirmed. Interestingly, the majority of differentially expressed phosphorylated proteins observed were nuclear proteins. Three regulators of apoptosis, Bad, Bax and Acinus, were also differentially phosphorylated in the two cell lines. Further development of this strategy will facilitate an understanding of the mechanisms involved in malignancy progression and other disease-related phenotypes.

Figure 1

Microarray strategy for global evaluation of phosphorylation changes as a function of disease

Figure 2

2D liquid separation of pre-malignant AT1 and malignant CA1a cell lines. Each lane represents a pH fraction different by 0.2 units. Vertical axis refers to the retention time during the separation. Intensity of the bands correspond to peak heights which ranged from a value of 100 mV to 990 mV. Difference between premalignant and malignant sample appears in the middle panel

Figure 3

Detecting phosphoproteins on microarrays using ProQ Diamond dye and anti-phosphotyrosine antibodies. (a) A study done with standards where ovalbumin, B-casein and a mixture of tyrosine phosphorylated proteins were used. Notice that when probed with both ProQ and anti pY antibody, solely pY proteins appear red, mixture of pY and pS or pT appear yellow and solely pS or pT appear green. (b) An image of a section of a protein microarray containing fractionated proteins from a malignant breast whole cell lysate.

Figure 4

Comprehensive study to assess reproducibility of the method. (a) CF chromatograms of 3 ca1a separations are shown on the left and of 2 AT1 separations are shown on the right. In all cases 4.5 mg of sample was loaded (one AT1 separation was performed with only 3 mg of total protein). Co-plotted with the chromatograms are pH profiles to illustrate that the pH gradient was consistent in all separations. (b) 2^nd dimension chromatograms of all batches of cell lines for pH ranges 6.4−6.2 and 7.2−7.0. Arrows along the chromatogram illustrate peaks that are shown in subsequent microarray data. (c) Array images of samples from pH fractions 7.2−7.0, 6.4−6.2 and 5.4−5.2 to illustrate reproducibility throughout the separation space. (d) Sections of microarray data showing an example of reproducible positive spots that are unique to ca1a (pH 5.4−5.2, retention time 28 min) and that are found in all cell lines (pH 6.4−6.2, retention time 26 min). Peaks corresponding to the positive spots found in all cell lines are indicated by arrows in fig 2b.

Figure 5

Selected microarray images showing comparison of spots where differential phosphorylation was observed between pre-malignant and malignant breast cell lines over different pH regions. Protein IDs as determined by tandem mass spectrometry are shown beside the image. For some proteins, multiple consecutive spots light up due to diffusional broadening during peak collection. In some cases more than one phosphoprotein was identified in the same collected fraction. In such cases, both phosphoproteins are listed.

Figure 6

Pie chart illustrating subcellular location of phosphoproteins whose phosphorylation sites were confirmed by mass spectrometry in both the AT1 and CA1a cell line combined. Closer examination showed a majority of these phosphoproteins to be present in the CA1a cell line (see table 1).

Figure

7: Functional classification of proteins differentially phosphorylated in the pre-malignant and malignant breast cell lines. Majority of phosphoproteins were found in the malignant, CA1a. In cases where a phosphoprotein was found in AT1 and not CA1a, it appears in a box with broken lines.

Figure 8

(a) Tandem mass spectrum (with +1 ion series highlighted) of selected phosphopeptide from apoptotic condensation inducing factor with inset showing phosphorylation difference between AT1 (boxed in red) and CA1a (boxed in blue) as seen on the microarray. (b) Microarray image together with complementary portion of reverse phase chromatogram where 60S ribosomal protein L14 was found to be phosphorylated in only CA1a.

Figure 9

Comparison of reversed phase chromatograms of pH fraction 7.0−6.8 from AT1 and CA1a to illustrate presence of larger amounts of transcriptional and translational regulatory proteins in malignant CA1a but not in pre-malignant AT1.