in the field of proteomics / peptidomics mass spectrometry has become a well - established tool for protein / peptide sequencing [ 13 ] . 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 / 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 , 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 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 . 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 ) and on - chip temperature control [ 9 , 10 ] . as a possible application of this system the analysis of cell - to - cell communication in saccharomyces cerevisiae it is long known that peptides play an important role in cell - to - cell communication in yeast cultures . 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 . 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 . a wt saccharomyces cerevisiae strain ( cen.pk 113 - 1a ) mating - type alpha was used . cells were grown in mineral medium ( mm ) with addition of glucose ( 2% ,  w / v ) as sole carbon source . prior to the transfer into the actual analysis chamber , yeast cells were precultivated in shaker flasks ( 10  ml  mm , 30c , 200  rpm ) . such 24-hour culture has a typical optical density of 19 - 20 at a measuring wavelength of 600  nm ( od600 ) . dilution to an od600 of 0.1 yielded the initial cell density chosen for all experiments . for every analysis , 1  ml of initial culture was transferred into a custom modified 2  ml glass vial which was prepared for connection to the analysis system . in the protease inhibition experiments , 10  m pepstatin ( sigma aldrich ) was added to the culture flask after precultivation . a modified 2  ml glass vial with cap including septum ( agilent technologies , usa ) was used as a basic module for the miniaturized cell culture chamber . fused silica tubings ( inner diameter 100  m ; bgb analytik ag , switzerland ) were inserted through the perforated septum , to provide two liquid in- and three liquid outlets ( figure 1 ) . all tubings inside the vial were fitted with a porous glass ending to allow cell culture medium to pass through , while preventing cells to leave the vial and enter the analysis system . airtight closure of the vial , essential for the functionality of the system , was achieved by deposition of a silicone rubber - based sealant ( bison , netherlands ) on top of the cap . to obtain efficient mixing of the cell suspension the cell culture chamber was equipped with a small magnet and kept on a magnetic stirrer ( 500  rpm ) . sampling of supernatant from the culture was done by creating an overpressure inside the vial . one of two inlets (  pressure  , figure 1 ) was connected to a pressurized air system . the second inlet (  mm  , figure 1 ) was connected to the medium reservoir ( via syringe pump 1 , sp1 , figure 2 ) for a constant supply of fresh mineral medium ( mm , 0.5  l / min ) . in operation only one of the three outlets (  sampling  , figure 1 ) was opened at a time , with the overpressure inside the chamber resulting in a steady sampling of supernatant . the complete system consists of the cell culture chamber placed on a magnetic stirrer ( ika labortechnik , germany ) , two syringe pumps ( fusion 200 ; chemyx , usa ) , a six - port valve ( rheodyne , usa ) controlled by an external interface , three capillary columns packed with 5  m silica - based c4 beads ( 300   pore size , reprosil ; dr . maisch gmbh , germany ) for peptide enrichment / concentration and pressure stabilization , an electrospray unit for sample deposition , a maldi target plate , and an x - y - z motion controller ( mm4005 ; newport , usa ; figure 2 ) . the syringe pumps supply solvent for elution ( sp2 ) and mm ( sp1 ) , keeping the volume in the vial constant . in the current experiments , no additional glucose or vitamins an in - house software program was developed and loaded into the microcontroller of the mm4005 for synchronization with the six - port valve . real - time determination of optical density was realized with a fiber spectrophotometer ( avaspec-2048 ) and suitable light source ( dh-2000 ; avantes , netherlands ) . ms analysis was performed directly from the spotted samples in a maldi q - tof mass spectrometer ( qtof premier ; waters , manchester , uk ) , equipped with a solid state ndyag laser . the optical density was measured with a fiber spectrophotometer setup especially conceived for use with 2  ml glass vials . initial linearity of the device at 600  nm was established for ods between 0.1 up to 1.5 . an extended calibration curve was recorded to get valid data at higher densities as well . after averaging of more than 200 individual measurements for one data point and curve fitting ( matlab , the mathworks , usa ) linearity was obtained for values up to od600 13 , fully covering the range of interest ( figure 3 ) . operation of the sampling system was basically divided into two parts : ( i ) sample accumulation / concentration and ( ii ) elution . keeping in mind the life cycle of s. cerevisiae ( reproduction / cell division each 75120 minutes ) this resolution gives chronological information about the state the whole culture is going through rather than information at the single - cell level . in the accumulation step ( 90  min ) the analysis column c1 was connected directly with the open outlet of the cell culture chamber via valve v1 ( figure 2 ) . the outflow of solution in that time was spotted onto a waste position on the maldi plate . the flow was adjusted to 0.40.6  l / min resulting in a sampling volume of 36 to 54  l over the 90 minutes accumulation / concentration period . during this step solvent was pumped through column c2 ( figure 2 ) connected via the valve to waste . the inclusion of column c2 proved necessary for maintaining constant backpressure inside the system and hence constant ( out- ) flow . after accumulation , elution followed by switching the valve , which simultaneously triggered the motion controller to position the first spot on the maldi plate exactly under the electrospray unit . solvent ( water / acetonitrile / acetic acid ; 10  :  90  :  0.6 ,  v / v / v ) was pumped through the column c1 at a flow rate of 1  l / min . eluates were deposited for 1.5  min per spot ( corresponding to 1.5  l ) . ten sample spots were collected in a row to ensure complete elution of the column . carryover between consecutive analysis runs can be excluded as empty ms spectra ( no peptide ion peaks ) were obtained for the last sample spots of each series . extraction of supernatant out of the cell culture chamber continued during the elution step as well . the sample was continuously pushed through a second waste column ( c3 , figure 2 ) for flow stabilization reasons . both , accumulation and elution step , were repeated up to 11 times , equivalent to more than 19 hours of total analysis time . throughout the experiment the optical density was measured at a 2 hours interval , and the resulting growth curve was recorded ( see e.g. , figure 5 ) . prior to sample spotting , the maldi plate was ultrasonically cleaned in ammonium bicarbonate solution ( 10  mm ) followed by water / methanol / trifluoroacetic acid ( 50/50/0.1 , v / v / v ) . alpha - cyano-4-hydroxycinnamic acid was used as matrix ( dissolved at 6  mg / ml in water / acetonitrile / trifluoroacetic acid ; 50/50/0.1 , v / v / v ) . after electrospray deposition of the samples , 0.8  l of matrix solution was added to each spot on the maldi plate . direct connection of the presented setup to an esi ms instrument is feasible but requires both parts , cell cultivation as well as sample analysis , to be physically linked which prohibits independent operation and requires all instruments to be placed at the same location . alpha - pheromone ( trphistrpleuglnleulysproglyglnpromettyr , monoisotopic mass 1682.84  da ) is detected in our maldi q - tof ms as [ m + h ] at m / z 1683.85 . associated with this ion often a peak at m / z 1699.84 is observed , corresponding to the peptide oxidized at position met12 ( a very common posttranslational modification ( ptm ) ) . the identity of the peptide could be confirmed by cid of the 1699.84 precursor ion ( ms / ms spectrum given in figure 4 ) . the alpha - mating - factor - related peptides were detected nearly throughout the whole analysis indicating that alpha factor is expressed and secreted constitutively ( also in the absence of opposing - mating - type cells / pheromone ) . the appearance of alpha - mating factor was more obvious at the late exponential growth phase ( corresponding to the diauxic shift , when conditions get less favorable , after nutrient consumption ) . two major fragments of the alpha - mating factor ( the aminoterminal hexapeptide and the carboxyterminal heptapeptide ) were detected in the medium ( verified by ms / ms , data not shown ) besides the intact pheromone . these two mating - factor ( degradation- ) fragments may be products of a protease which cleaves the intact pheromone in two pieces ( see section 3.4 ) . the fact that their amounts increase in time while intact pheromone decreases at later stages of growth would agree with this . the objective of this study was to develop an automated setup for the analysis of conditioned media of saccharomyces cerevisiae cultures at different growth stages at a miniature scale . to obtain reproducible results and to allow valid comparisons between experiments , it is important to keep the cells at the same physiological / growth state for all experiments . for this a fiber spectrophotometer was integrated in our setup , specifically designed for use with 2  ml glass vials . this allowed real - time noninvasive determination of culture ods . to cover the whole range of ods a typical yeast culture under the applied conditions goes through ( 0.1 to 13 ) , the real - time recorded growth curves were accurate , as they were in excellent accordance with calibration curves obtained from standard od determination techniques ( diluting the culture to ods in the linear range of the spectrophotometer and recalculating the actual od , data not shown ) . our analyses confirmed that different batches of yeast cultures show very similar growth behaviors ( figure 5 ) . however , for a meaningful comparison between cultures at various time points / growth stages a perfect match of the two curves is essential . parameters like small variations in the initial ods of the inoculated culture or the addition of a test compound may result in slightly delayed or shifted initiation of cell growth . this can be corrected for by software - wise adjustment (  warping  ) of one of the two curves onto the other ( a simple shift along the time axis often being sufficient ) . the finally obtained diagram shows a perfect match in terms of cell growth of both investigated conditions ( figure 6 ) . all timepoints of both series overlap on a single curve which facilitates a valid comparison . to study the cell culture medium during a standard yeast growth , samples were collected at 2 hours intervals starting at an od600 of approx . 1 , which typically is reached 18 to 20 hours after inoculation of the initial culture ( od600 of 0.1 ) . up to 11 consecutive samples were analyzed by maldi q - tof ms(/ms ) yielding chronologically classified ms spectra (  peptide profiles  ) . figure 7 shows representative spectra at every second time point sampled ( resulting in a difference of 4 hours between each consecutive spectrum displayed ) . several different peptide - like signals are evident in the mass spectra acquired . to assist in the interpretation of these profiles , 5 mass over charge ( m / z ) values of interest are highlighted throughout all the spectra , ( four between 1520 and 1720  m / z and one at about half the  m / z ) . ms / ms analysis confirmed that the peaks at m / z 1683.85 , 1699.83 , and 1536.75 represent the mating factor , its oxidized version , and a c terminally truncated species ( loss of tyr residue ) , respectively . the peak in the lower m / z region , at 882.45 , represents one of the two mating - factor cleavage products ( the aminoterminal hexapeptide ) . one of the most distinct peptide peaks shows up at 1628.74  da ( figure 7 ) . ms / ms analysis and mascot database searching ( using  no enzyme  as parameter ) identified it as the soluble fragment of a cell wall protein ; exo-1,3-beta - glucanase ( exg1 ) . exg1 is known to be involved in cell wall organization by enabling beta - glucan assembly . literature data   and our time - course analysis confirm its presence in the culture medium just before alpha - mating - factor secretion . this suggests that this protein fragment is shed from the membrane just prior to , or simultaneously with , mating - factor release , which may imply a potential role of this protein fragment in mating . for the time - resolved detection of mating - factor , cells were grown in mm without addition of any special component except those needed for cell growth . it was observed that none of the four mating - factor - related peaks appear early during growth . first unequivocal detection is between hour 26 and 28 , that is , at an od600 of approx . it is remarkable that both intact mating factor and one of its fragments ( the aminoterminal hexapeptide ) are detected virtually simultaneously . figure 7 should be understood as an illustrative sequence of ms spectra of a single continuous ( 40 hours ) analysis run . to illustrate the usefulness of the miniature culture media analysis system , a simple experiment was designed looking at the effect of a protease inhibitor on the appearance of selected peptide fragments detected above . the proteolytic cleavage of mating factor at the leu / lys peptide bond suggests involvement of an aspartic protease . hence a general aspartic protease inhibitor was selected to study its effect on the appearance of the peptides / peptide fragments observed . the anticipated effect reduced appearance of the 881.45  da fragment ( [ m + h ] at m / z 882.45 ) during the whole experiment . besides the addition of 10  m of pepstatin all conditions were kept strictly the same as for the other experiments . the first appearance of intact mating factor and its oxidized variant was observed at the same time point as for the culture without inhibitor . this indicates that the addition of pepstatin at the chosen concentration had no effect on the actual secretion of alpha factor ( ms data not shown ) . the abundance of the 881.45  da fragment on the other hand was significantly lower in all spectra ( figure 8 , right scale ) . the red , dashed line in figure 8 ( left scale ) depicts the growth curve ( in terms of optical density ( light absorbance ) at 600  nm ) at the time of sampling . values plotted are representative as biological repetitions of the experiment over shorter time frames showed the very same trend . during the yeast life cycle , mating factor is the trigger for two haploid cells of opposite mating type ( alpha and a ) to mate and form one single diploid cell . this happens in nature once the growth conditions get unfavorable , for example , lack of nutrition . however , it is clear that these haploid cells still produce their pheromone in the absence of an opposing mating type or pheromone . signal for mating  particularly at the later stages of growth , that is , at the end of and after the exponential growth phase . in the early stages of growth no peaks representing the mating factor were identified . comparing the growth curves of the pepstatin containing with those of cultures confirmed that the growth behavior of the cells was similar in both conditions , justifying a valid time - based peptide profile comparison ( figure 6 ) . we hypothesized that if mating factor is secreted already at an earlier cell growth stage but readily cleaved by a protease , inhibition or inactivation of this protease could promote the detection of intact mating factor at an earlier time point in the growth curve . however , it should be noted that the time resolution of the current setup was 2 hours . the comparison of cell cultures with and without pepstatin showed significant differences in terms of the extracellular peptide profiles . the presumed aspartic protease responsible for the formation of the 881.45  da fragment clearly seems to be inhibited by pepstatin . at all examined time points the ratio between the overall count of the 881.45  da fragment and that of the intact mating factor ( both native ( 1682.84  da ) and oxidized ( 1698.83  da ) ) was significantly decreased in the culture containing the protease inhibitor ( figure 8) . only at the latest points of inspection , the stationary stage of cell growth , a noticeable count of the 881.45  da fragment was detected but still far below the intensity level of that in the pepstatin - free culture . given that the conditions for both cultures were kept identical , the disappearance or drastic reduction of the fragment in the extracellular medium is clearly to be attributed primarily to the addition of pepstatin . this suggests that pepstatin inhibits the potential protease responsible for  normal  alpha - mating - factor peptide cutting . in the obtained ms spectra additional mating - factor - related ions the detection of oxidized mating factor missing the tyrosine at the c - terminus at the later growth stages ( m / z 1536.75 ; figure 7 ) may reflect the action of an ( carboxyterminal ) exopeptidase in the extracellular medium . concurrently , the amount of intact mating factor inside the culture decreases . besides ions related to mating factor , another peptide possibly involved in the secretion process was identified ( m / z 1628.74 ; figure 7 ) . database searching identified it as the soluble part of the exo - b-1,3-glucanase exg1 ( a cell wall protein ) . the role of this protein in peptide secretion remains elusive , but comparing its appearance in both experiments showed marked differences . in standard cultures , the peptide was found most abundant prior to secretion of mating factor and slightly reduced at later growth stage . in - pepstatin containing cultures this peptide was not found prior to mating peptide secretion , and it appeared considerably less prominently present at later growth stages as well ( data not shown ) . the presented automated system allows in - line sampling of microliter amounts of extracellular conditioned cell culture media , preparing them for maldi ms analysis . the minimal amount of cell culture required for this has advantages in terms of handling and cost reduction . for example , compared to standard flask cultivation , an enzyme inhibition study during cell growth could be completed with 10 times less amount of the commercial compound ( i.c . in particular when effects on cultured cells of more expensive compounds have to be tested , the experiment cost savings related to the reduced culture chamber volume will become more substantial . implementation of the real - time optical density measurement in - line ( without disturbing the cell culture ) made many tedious extra sample collection and dilution steps redundant and resulted in an overall increase of the practicability of the system . in summary this reduces the risk for losses of peptides sticking to microtip or tubing / column wall materials , which often drastically reduces the overall sensitivity of the analysis .
we present a fully automated setup for performing in - line mass spectrometry ( ms ) analysis of 
 conditioned media in cell cultures , in particular focusing on the peptides therein . 
 the goal is to 
 assess peptides secreted by cells in different culture conditions . 
 the developed system is 
 compatible with ms as analytical technique , as this is one of the most powerful analysis methods 
 for peptide detection and identification . 
 proof of concept was achieved using the well - known 
 mating - factor signaling in baker 's yeast , saccharomyces cerevisiae . 
 our concept system holds 1 
 ml of cell culture medium and allows maintaining a yeast culture for , at least , 40 hours with 
 continuous supernatant extraction ( and medium replenishing ) . 
 the device 's small dimensions 
 result in reduced costs for reagents and open perspectives towards full integration on - chip . 
 experimental data that can be obtained are time - resolved peptide profiles in a yeast culture , 
 including information about the appearance of mating - factor - related peptides . 
 we emphasize that 
 the system operates without any manual intervention or pipetting steps , which allows for an 
 improved overall sensitivity compared to non - automated alternatives . 
 ms data confirmed 
 previously reported aspects of the physiology of the yeast - mating process . 
 moreover , matingfactor 
 breakdown products ( as well as evidence for a potentially responsible protease ) were 
 found .