In the field of proteomics/peptidomics mass spectrometry has become a well-established tool for protein/peptide sequencing [1
]. Its steadily increasing performance (sensitivity as well as resolution) enables the analysis of thousands of different molecules at the same time which is of big advantage for “shotgun” approaches, where complex mixtures of unknown samples are targeted for identification. In combination with sophisticated separation methods, protein/peptide analysis has become much faster and more efficient [4
Nevertheless, the whole analysis cycle, starting with peptide extraction from the medium of interest, sample pretreatments (chromatographic purification, digestion) prior to the ultimate injection in the MS instrument requires many time consuming and tedious steps, often done manually. Furthermore, the need for pipetting induces unavoidable sample losses, resulting in a decrease of the overall method sensitivity.
The goal of this work was the design and realization of a system, capable of performing sample extraction, protein/peptide enrichment, purification, and sample preparation for MALDI MS analysis in a fully automated and controlled manner. With the elimination of all previously necessary sample handling steps requiring pipetting, the sensitivity achievable by the system is boosted. Furthermore, using MALDI MS instead of direct connection to an ESI instrument allows for decoupling of the cell cultivation and the actual sample analysis. In that way those two parts can be performed independently from each other, even at different locations.
In addition, sample volumes are kept at a minimum. Reasons to pursue miniaturization include reagent costs. In many studies, different chemicals or additives are needed at certain concentrations to reveal activities of different components in the cell culture. The investment for additives is evidently reduced if the total sample volume is small. Besides these “economy” factors, evolution towards microscale is an essential step to a possible future design of a fully integrated, on-chip analysis system [7
]. Once integrated on a single chip, all the advantages of those can be exploited, including (but not limited to) faster analysis cycles, implementation of extrasensing elements (e.g., viability analysis [8
]) and on-chip temperature control [9
As a possible application of this system the analysis of cell-to-cell communication in Saccharomyces cerevisiae
(baker's yeast) cultures based on peptide secretion was investigated. It is long known that peptides play an important role in cell-to-cell communication in yeast cultures [11
]. As best documented example, we selected the mating process as model to evaluate the performance of our novel system. During mating, two yeast cells of opposite haplotype secrete a 13 amino acid pheromone called alpha-mating factor (secreted by alpha-type cells) and a 12 amino acid residue a-mating factor (released by a-haplotypes), respectively. This initiates alpha- and a-haplotype cell fusion to form a diploid cell [12
]. In the course of this study the focus was on the detection, accumulation, and analysis of this peptide at different stages during cell culture growth. Therefore, cells were cultivated at small scale (1
mL) while continuously extracting and analyzing the extracellular conditioned medium. As a result a chronological sequence of MS spectra was obtained that could be nicely correlated to the corresponding growth stages. In a second study the effects of an enzyme inhibitor on potential peptidase activity cleaving alpha-mating factor was investigated. This experiment enabled us to collect evidence supporting the hypothesis for the involvement of a yapsin-like protease in an easy and fast way [13