Comparative proteomic methods are rapidly being applied to many different biological systems including complex tissues. One pitfall of these methods is that in some cases, such as oncology and neuroscience, tissue complexity requires isolation of specific cell types and sample is limited. Laser microdissection (LMD) is commonly used for obtaining such samples for proteomic studies. We have combined LMD with sensitive thiol-reactive saturation dye labelling of protein samples and 2-D DIGE to identify protein changes in a test system, the isolated CA1 pyramidal neurone layer of a transgenic (Tg) rat carrying a human amyloid precursor protein transgene. Saturation dye labelling proved to be extremely sensitive with a spot map of over 5,000 proteins being readily produced from 5 mug total protein, with over 100 proteins being significantly altered at p < 0.0005. Of the proteins identified, all showed coherent changes associated with transgene expression. It was, however, difficult to identify significantly different proteins using PMF and MALDI-TOF on gels containing less than 500 mug total protein. The use of saturation dye labelling of limiting samples will therefore require the use of highly sensitive MS techniques to identify the significantly altered proteins isolated using methods such as LMD.

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During lactation mammary cell number is regulated by an intramammary mechanism sensitive to the frequency of milking. Thus, a unilateral increase in cell number is elicited when one gland of lactating goats is milked thrice instead of twice daily [l]. The local increase in cell number i s associated with a higher rate of DNA synthesis in the thrice-milked gland [l]. However, it is also possible that the response is due in part to a reduction in the loss of mammary secretory cells at this stage of lactation. Since the effect i s related to frequency of milk removal from the gland we wished to determine whether goat's milk contains factors which may act as local regulators of mammary cell number. In this study we used mammary cells embedded within collagen gels, a culture system which allows extensive cell proliferation and formation of three-dimensional outgrowths reminiscent of the tissue in vivo [2].Mammary cells prepared from late pregnant mice by collagenase digestion [3] were fractionated on a Percoll gradient. Epithelial cells were suspended in foetal calf serum containing 10% DMSO and stored frozen under liquid N,. A batch of cells was thawed for each experiment. Cells were suspended at 4°C and lo6 cells/ml in rat tail collagen solution (1.6 mg/ml, pH 7.4), and aliquants (1.5 ml) were immediately gelled in 3.5 cm diameter wells at 37°C. Cells were cultured for 2-8 days in medium 199 / Hams F12 (5050) containing 15 mM-Hepes, 10% foetal calf serum, 5 pg insulin/ml, 1 pg hydrocortisoneiml, 0.65 ng triiodothyronine/ml, 10 pg epidermal growth factor/ml, 0.12 pg prolactidml, 27 pg oestradiol/ml and 0.78 ng progesterone/ml, and in the presence or absence of a milk fat globule membrane (MFGM) fraction prepared from goat's milk. Milk fat obtained by centrifugation (2500g, 20 min, 20°C) was vortex-mixed and membranes were sedimented by centrifugation at 100,OOOg for 90 min [4]. The MFGM fraction was incubated in 0.3 pM-glutathione pH 6.0 at 37°C for 24 h , centrifuged again, and the supernatant was dialysed against water and freeze-dried. MFGM was added to culture medium (3 ml/well) at twice its milk concentration, and medium was changed every 2 days. DNA synthesis was measured on day 6 and 8 by incorporation of [methyl-'H]thymidine (2 pCi/ml) during the last 4 h of culture, and cells were harvested by collagenase digestion for assay of DNA content [5].

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