cysteine - rich peptides are of significant and growing interest due to the increasing use of tetracysteine tags for fluorescence imaging of living cells(1 ) and as tools for bioremediation and phytoremediation of toxic heavy metal contaminants such as cadmium and mercury.(2 ) a library of 205 designed tetracysteine peptides immobilized on cellulose has been successfully screened against biarsenical probes.(3 ) but , the drawback is that these spatially addressed libraries tend to be orders of magnitude smaller than biological or bead - based libraries . cell - based cysteine - rich libraries have also been successfully used to identify optimized peptide tags for biarsenicals.(4 ) however , cytotoxicity is an intrinsic liability in the use of cell - based libraries to identify peptides that can sequester heavy metals . in contrast , phage libraries can be screened under abiotic conditions and are less susceptible to cytotoxic effects . they have been used to identify peptides that bind to immobilized metals , but immobilized metals present few open coordination sites for tight peptide binding of phage - displayed peptides.(5 ) a complementary method is needed for identification of cysteine - rich peptides that tightly bind to free metals and cytotoxic ligands . one - bead one - compound ( oboc ) peptide libraries are powerful tools to simultaneously screen millions of peptides for exceptional reactivity and selectivity.(6 ) they can incorporate both ribosomal and nonribosomal amino acids and can be screened under conditions toxic to cells . however , due to the problems caused by cysteine during synthesis , screening , and sequencing , cysteine is generally excluded from oboc libraries . standard conditions ( 1:1 dmf / ch2cl2)(7 ) for the synthesis of polycysteine peptides using tritylcysteine are inefficient due to the bulk and hydrophobicity of the s - trityl group , leading to aggregation and ultimately to racemization . alternative protecting groups such as s - acetamidomethyl and s - t - butylthiocysteine can be used to deter aggregation , but at the cost of reagent toxicity , additional side reactions , and additional deprotection steps . thiols will form disulfides under ambient conditions , so screening and sequencing must be performed under reducing conditions to prevent disulfide formation . free cysteine thiols can undergo side reactions during edman degradation and during cyanogen bromide cleavage  chemistry used in the two traditional methods of direct sequencing of oboc - derived peptides . in addition , cysteine can be difficult to distinguish from arginine by edman degradation.(8 ) the first cysteine - rich oboc library has recently appeared in the literature.(9 ) while this library was neither screened nor sequenced , randomly chosen metal - soaked beads were extracted and analyzed to reveal interesting variations in metal affinities . clearly , a robust method for sequencing cysteine - rich oboc libraries would be invaluable . a wide range of methods are available to identify the sequences of peptides on single beads . after screening an oboc library and isolating bead hits , there are either indirect or direct approaches to identify the sequences of these library hits . the indirect approaches like recursive deconvolution are laborious while the use of encoding tags introduces various complications.(10 ) the direct sequencing of individual beads from oboc peptide libraries traditionally uses edman degradation(6 ) which is restricted to -amino acids(11 ) and requires access to a free n - terminus . surprisingly , after two decades of research using oboc peptide libraries , free cysteines have been included in variable positions and identified by edman sequencing in only a handful of instances.(12 ) continuing advances in the efficiency and economy of mass spectrometry have led to powerful new methods of peptide sequencing based on chemical formation of mass ladders . tandem mass spectrometry ( ms / ms)(15 ) is still the most direct approach to peptide sequence determination and does not share the structural limitations imposed by edman chemistry.(16 ) therefore it is suprising that there are so few examples of ms / ms sequencing applied to peptides obtained from single beads . in no case have single - bead ms / ms analyses been applied to oligomers containing cysteine . despite the lack of precedence , we chose to use ms / ms as our method to sequence thiol - rich compounds from a highly diverse oboc library in order to reduce postscreening chemical manipulations and facilitate compatibility with capped n - termini , beta peptides , and peptoids.(19 ) a wide range of major and minor problems conspire to make the synthesis , screening , and sequencing of cysteine - rich oboc peptide libraries , at best , impractical and , at worst , impossible , using any combination of existing methods . herein we describe the first synthesis , screening , and direct sequencing of a cysteine - rich oboc library and validate this methodology by screening for and discovering peptides that form highly fluorescent complexes with the biarsenical flashedt2 . therefore , we first developed robust conditions for the synthesis of a cysteine - rich peptide library and optimized the cleavage and sequencing of peptides from individual beads from a cysteine - rich combinatorial library . we then screened a cysteine - rich library in the presence of thiol and phosphine reducing agents to identify optimal ligands for the biarsenical flashedt2 . cysteine is often incorporated into peptides with an s - acetamidomethyl protecting group that can be removed using mercuric acetate . however , mercury is difficult to remove from cysteine - rich peptides(20 ) and can lead to beta elimination , generating dehydroalanine intermediates.(21 ) indeed , removal of acetamidomethyl groups from polycysteines like ac - ccpkwccckkm - nh2 using mild conditions ( 60 mm hg(oac)2 in 1:1 acoh / dmf ) led to low yields due to formation of dehydroalanines which were apparent in the mass spectra of the products . the problems of mercury deprotection have been overcome in the synthesis of ac-(-glu - cys)2gly - nh2 using s - t - butylthiocysteine , but it required an additional deprotection step.(22 ) tritylcysteine is a good alternative for incorporation of cysteine since the s - trityl protecting group is removed under standard acidic deprotection conditions . however , the standard conditions ( 1:1 dmf / ch2cl2)(23 ) for the synthesis of polycysteine peptides using tritylcysteine is inefficient due to the bulk and hydrophobicity of the s - trityl group , leading to aggregation and ultimately to racemization.(24 ) through experimentation , we found that using nmp as the solvent for all couplings during synthesis of polycysteines alleviates the problems with tritylcysteine . as a prelude to construction and screening of a cysteine - rich library it was essential to demonstrate that we could cleave cysteine - rich sequences from tentagel using cyanogen bromide . we synthesized a biarsenical - binding tetracysteine peptide ( ac - wdccpgcckm)(1 ) on tentagel in order to optimize conditions for cyanogen bromide cleavage . the first problem we encountered was centered on the reactivity of the cysteine thiols . while there is precedent for chemoselective cyanogen bromide cleavage of full - length proteins containing cysteine,(25 ) we found that cyanogen bromide cleavage of the cysteine - rich peptide led to a wide range of side products ( figure 1 ) , presumably due to thiocyanate and disulfide formation . to overcome these problems with cyanogen bromide cleavage , we first treated the beads with dithiothreitol ( dtt ) and then alkylated the cysteine thiols with iodoacetamide in a sodium carbonate buffer . these extra steps led to a dramatic improvement in the purity of the cleavage products ( figure 2 ) . lc - ms analysis of the crude products of cyanogen bromide cleavage of ac - wdccpgcckm from tentagel . lc - ms analysis of the crude products of cyanogen bromide cleavage of ac - wdc*c*pgc*c*km from tentagel . the alkylation / cleavage chemistry was readily applied to individual beads in millipore ultrafree - mc centrifugal filter units adapted for microcentrifuge tubes . unfortunately , single beads are poorly solvated in the alkylation step and often float at the aqueousair interface , leading to incomplete reduction and alkylation . to ensure reproducibility , we included methanol as a cosolvent in the alkylation procedure . under these conditions the beads are well - solvated and sink to the bottom of the tube , leading to clean , reproducible alkylation . a cysteine - rich decapeptide library ( ac - x8-km - tentagel ) with a potential diversity of 18 ( all amino acids excluding methionine and isoleucine ) was synthesized on 5 g of 130 m tentagel ( approximately 6.5 million beads ) . ile was excluded because leu and ile are isobaric ( have exactly the same mass ) . lysine was included near the c - terminus to favor the formation of y - ions in the ms / ms spectrum of the doubly charged peptide . the library was biased toward cysteine by coupling half of the resin to fmoc - tritylcysteine during each round of coupling . fourteen beads were selected at random from the library and subjected to the cysteine alkylation and cyanogen bromide cleavage conditions . the samples were then subjected to nanoflow lc - ms / ms for sequencing of the doubly charged molecular ions . for these cleaved peptides , which are different from traditional tryptic peptides , manual interpretation of the ms / ms spectra proved to be more reliable than automated interpretation . the ms / ms for the peptide cleaved from bead cys01 ( figure 3 ) , and all beads from this library , exhibits a peak at m / z 230 corresponding to the y2-ion derived from the c - terminal lys - hsl . using m / z 230 as starting point one can readily compare successive peaks to a table of potential y3-ions.(26 ) by continuing this style of analysis the remaining y - ions were identified . the ms / ms of the peptide cleaved from cys01 also exhibits a strong signal at m / z 203 , corresponding to the b1 ion derived from n - terminal ac - cys*. in cases where the y - series of ions was incomplete , b - ions could resolve ambiguities near the n - terminus . of the 14 crude cleavage samples , four did not exhibit a significant doubly charged ion required for good ms / ms fragmentation . ten of the samples exhibited a doubly charged molecular ion amenable to sequencing , and nine of these samples yielded unambiguous sequences for cys01cys09 ( table 1 ) . the main product from bead cys06 was found to be a truncated peptide , probably due to a problematic coupling step during synthesis . the peptide from cys10 did not yield a complete set of y - ions and could not be fully sequenced . ultimately , with this challenging class of polycysteine - containing peptides , 60% of the samples yielded complete sequences ( cys01cys09 ) . for comparison , beads from a library of cysteine - free peptides ( ac - kx8km - tentagel ; x  c , m , i ) , which were not subjected to reduction and alkylation , were found to yield unambiguous sequence data in 6 out of 8 ( 75% ) cases . tsien and co - workers evaluated series of peptide ligands for biarsenicals and concluded that the ccxxcc hexapeptide motif is optimal for binding and fluorescence of the biarsenical flash . subsequently , a mammalian cell library and a cellulose array were used to identify optimized ccxxcc tetracysteine peptide tags for flash . two types of optimized tetracysteine motifs appear to be superior to all others : ccpgcc and cc(k / r)xcc.(3 ) the proven superiority of these flash tags provided us with the opportunity to validate our protocols for screening and sequencing of our cysteine - rich library . our library was heavily biased toward cysteine by coupling 50% of the resin to cysteine at each stage of the library synthesis . this kind of user - control over amino acid composition is difficult to achieve with biological libraries synthesized on a dna synthesizer , but is easy to achieve with oboc peptide libraries . while the potential diversity of our library ( ac - x8 km ; x  met , ile ) is around 10 billion ( 18 ) , the actual diversity of our library was limited by the amount of resin used ( about 6.5 million beads ) and the fact that 50% cysteine was incorporated at each and every position . this amount of resin does not fully cover the potential sequence space , but the cysteine bias optimizes the coverage toward known tetracysteine sequences . in order to ensure the presence of an optimized flash sequence ( wdccpgcck),(27 ) three beads with the known biarsenical tag ac - wdccpgcckm were added to the library . the beads ( 0.33 g resin ) were suspended in 50 ml of a low sodium / high potassium ph 7.2 phosphate buffer that approximates the ion content of mammalian cytoplasm.(28 ) several reducing agents , commonly used in cell staining with flash , were included in the screening buffer : beta - mercaptoethanol ( 1 mm ) , the bis - thiol bal(29 ) ( 0.5 mm ) , and tris - carboxyethylphosphine ( 2 mm ) . the library , containing about 130 mol total peptide , was exposed to 0.005 mol flashedt2 over 6 h. the beads were then washed with phosphate buffer containing triton x-100 . to increase the stringency of the assay , the beads were exposed to screening buffer containing 1 mm bal , but devoid of flashedt2 for 10 h. secondary thiol washes are a common method to improve contrast when staining mammalian cells with biarsenicals . the suspended beads were screened under a long wave uv lamp , which has sufficient output within the flash excitation range to readily identify hits . six beads exhibited strong to weak green fluorescence and were removed for sequencing ( figure 4 ) . each bead was then subjected to reduction , alkylation , and cleavage , and the cleaved peptide was analyzed by tandem mass spectrometry . three of the six cleaved peptides , fb1 , fb2 , and fb6 gave unambiguous sequences ( table 2 ) . the other three samples contained very little peptide , insufficient for ms / ms analysis , possibly due to interference with residual flash . to our satisfaction , one of the three sequences matched the sequence of the three beads doped into the library . surprisingly , this bead exhibited the weakest green fluorescence of the six beads selected for sequencing . since the fluorescence of bead fb6 was on the borderline of our criteria for selection , the other two doped beads may have been passed over . it is possible , but less likely , that the other two doped beads corresponded to the unsequencable samples fb3fb5 . all three of the flash - binding sequences contain a ccxxcc tetracysteine motif and suggest that it is superior to other short tetracysteine patterns : e.g. , cxcxcxc , cxxccxxc , etc . flash - binding peptide fb1 contains one of the two known efficient sequences ( cckxcc and ccpgcc ) ; peptide fb6 contains the other . peptide fb2 contains a potential flash tag that has not previously been identified : ccpccc . we have developed the first method for the synthesis , screening , and sequencing of cysteine - rich oboc peptide libraries . a diverse , heavily biased cysteine - rich library ac-(xxx)8lysmet ( xxx = 50% cys  met , ile ) was synthesized on tentagel resin with fmoc - tritylcysteine , using nmp to avoid tritylcysteine aggregation . conditions were developed for iodoacetamide alkylation and cyanogen bromide cleavage of single beads , and the samples were analyzed by tandem mass spectrometry . over half of the peptide samples cleaved from beads gave unambiguous sequences . cysteine - free peptides yielded slightly more unambiguous sequences than the cysteine - rich sequences . the method was validated by synthesizing and screening a cysteine - rich library against the profluorescent reagent flash . using this method , we successfully identified sequences containing two of the known short biarsenical tag motifs : ccpgcc and cckxcc . our synthesis and sequencing methodology expands the power of oboc libraries to include short , cysteine - rich peptides for applications such as biarsenical binding tags,(27 ) metal - chelating species,(9 ) and cysteine - rich pharmaceuticals.(30 ) all glassware used in the library synthesis was silylated with 5% dichlorodimethylsilane in dichloromethane , rinsed , and oven - dried . solutions of 0.5 m fmoc - amino acids and 0.5 m hbtu / hobt in nmp were prepared for the coupling reactions . beads were transferred between vessels with a silylated 10 ml electric pipettor as suspensions in nmp . the couplings were performed with 5 g of 130 m rapp polymer tentagel hl nh2 resin ( 0.45 mmol / g , batch no . 124.782 ) with 3 equiv of each amino acid , 3 equiv hbtu , 3 equiv hobt , and 6 equiv dipea in nmp . the first two residues ( met and lys(boc ) ) were sequentially coupled to the entire batch of tentagel in a 500 ml solid phase synthesis vessel . to ensure 50% incorporation of cysteine at each randomized position , one - half of the resin was coupled to fmoc - cys(trt)-oh in the 500 ml solid phase synthesis vessel ; the remaining resin was equally divided between 17 20 ml scintillation vials . the vials were gently agitated on a shaker during the coupling reaction . after coupling for 90 min , the resin was pooled in the spps vessel , rinsed with dmf ( 3  50 ml ) , and deprotected for 30 min with 50 ml of 20% piperidine in dmf . after the final amino acid was coupled , the n - terminus of each peptide in the library was acetylated in the spps vessel with 5 ml acetic anhydride and 24 ml dipea in nmp . the library was washed with dmf ( 3  50 ml ) , methanol ( 3  50 ml ) , and dichloromethane ( 3  50 ml ) . the protecting groups were removed by treatment of the resin with 94% tfa , 2.5% ethanedithiol , 2.5% h2o , and 1% triisopropylsilane for 3 h. following cleavage , the beads were washed with methanol ( 3  50 ml ) and ph 7.2 buffer ( 3  50 ml ) . the bead library was suspended in 50 ml ph 7.2 aqueous solution . to select random beads from the library , 200 l of suspended beads were transferred to a 90 mm petri dish using a p200 pipetman . individual beads were taken up with the p200 pipetman under a 50 dissecting microscope and transferred to a prewashed ( 2  250 l , methanol ) millipore ultrafree - mc spin tube with a 0.22 m durapore pvdf membrane . the bead was separated from solvent by centrifugal filtration by spinning at 12  000 rpm in a benchtop microcentrifuge . to avoid loss of the single bead , it is essential to confirm by microscope that the bead is at the bottom of the spin tube between washes . beads were subsequently washed with 30% aqueous methanol ( 2  100 l ) . small wash volumes and gentle agitation were used to confine the bead to the bottom of the spin filter . the cysteine sidechains of beads isolated from cysteine - containing libraries were reduced and alkylated to prevent side reactions during cyanogen bromide cleavage : to each tube was added 100 l of 50 mm dtt buffered with 0.4 m nahco3 in 30% aqueous methanol , and the tube was shaken for 1 h. next , 40 l of 0.5 m iodoacetamide in 30% aqueous methanol was added to each tube . after shaking for 30 min , 150 l of 50 mm dtt buffered with 0.4 m nahco3 in 30% aqueous methanol was added and the tube was shaken for 15 min . finally , the alkylation mixture was spun through the spin tube and the tube progresses to the rinsing phase . still inside of the spin tube , the bead was rinsed with 3:7 methanol/0.1% aqueous formic acid ( 2  100 l ) and 1:1 ch2cl2/meoh ( 2  100 l ) . the bead was then transferred as a suspension in 50 l meoh to a pcr tube . the meoh was evaporated under vacuum , leaving the bead in bottom of the tube . the presence and location of the bead was verified under the microscope . to each pcr tube was added 30 l of a solution of 20 mg / ml cyanogen bromide in 0.1 m hcl . each tube was covered from light and was then frozen at 78 c , and the solvent was removed by lyophilization . a solution of aqueous formic acid in acetonitrile ( 30 l of 1:1 ( v / v ) 0.1% formic acid / mecn ) was added to the pcr tubes , that were then vortexed for one minute to dissolve the residue . samples were analyzed on a waters qtof2 mass spectrometer in positive ion mode equipped with a waters nanoacquity hplc ( column : waters symmetry 100   100 mm c18 1.7 m particle size ) . doubly charged ions were selected and isolated for sequencing by survey scanning during chromatographic analyses . the flow rate was 350 nl / min ramped from 5% b to 80% b over 40 min . the electrospray voltage was 3 kv and the cone voltage 30 v. to assist nebulization , nitrogen gas was used at approximately 5 psi . argon was used as the collision gas at 4  10 torr on the analyzer s penning gauge . the ms / ms spectra were interpreted manually , primarily by assigning the peaks corresponding to the c - terminal y - ions . the y - ion peaks were abundant since every cleaved peptide had lyshsl ( hsl = homoserine lactone ) at the c - terminus . the interpretation of the spectra was simplified because the formation of b - ions was suppressed as a result of n - terminal acetylation . a cysteine - rich oboc library ( 0.33 g ; ac-(xxx)8lysmet ; xxx = 50% cys ;  met , ile ) was first prescreened to remove fluorescent beads . the beads were transferred to a 50 ml solid phase synthesis vessel . once inside the 50 ml vessel , the library was washed with 70:30 100 mm potassium bicarbonate / meoh ( 2  30 l ) . the beads were transferred to , and suspended in , a 90 mm petri dish using 30 ml of 100 mm potassium bicarbonate and observed in the dark under an 8 w long wave uv lamp ( spectroline enf-280c ) with a stereo microscope ( 1545 magnification ) . the library was then transferred back to the solid phase synthesis vessel for screenng using 100 mm potassium bicarbonate . at this point three beads bearing the sequence ac - wdccpgcckm , suspended in 10 ml deionized water , were added to the vessel using a 200 l pipetman . the library was then washed ( 3  50 ml ) with a potassium - rich phosphate buffer ( 15 mm nacl , 0.5 mm mgcl2 , 100 mm potassium phosphate , ph 7.2 ) containing 0.01% ( v / v ) triton x-100 . to the washed library in the solid phase synthesis vessel was added 50 ml of the potassium - rich phosphate buffer containing 0.03% ( v / v ) triton x-100 , 1 mm -mercaptoethanol , 0.5 mm bal , and 2.6 mm tris - carboxyethylphosphinehcl . flash - edt2 ( 5 l ) was added as a 1 mm stock solution in dmso . the vessel was then agitated on a shaker for 6 h. after 6 h , the screening solution was drained and the resin was extensively washed . the library was first washed with potassium - rich phosphate buffer containing 0.01% triton x-100 ( v / v ) ( 3  30 l ) . then , a phosphate buffer containing 0.03% ( v / v ) triton x-100 , 1 mm -mercaptoethanol , 1 mm bal , and 2.5 mm tris - carboxyethylphosphinehcl was added to the vessel , and the vessel was shaken for 10 h. after 10 h , the solution was drained and the resin was washed extensively with 100 mm potassium bicarbonate buffer ( 6  30 l ) until the odor of thiols from the resin was no longer diminished . finally , the library was suspended in 200 ml of 100 mm potassium bicarbonate buffer and divided equally into four 90 mm petri dishes for visualization / bead selection . each of the four library fractions 90 mm petri dish was observed under an 8 w long wave uv lamp , held 10 cm from the beads , using a stereo microscope to identify fluorescent beads . fluorescent beads were photographed with a nikon coolpix e4500 digital camera , top - mounted on the microscope ( exposure times between 1 and 8 s were used ) . a small amount of meoh was added by pipet to sink floating beads into the focal plane . after being photographed , the fluorescent beads were isolated from the surrounding nonfluorescent beads using the tip of a 200 l pipetman . once the surrounding beads were cleared , the fluorescent bead was drawn into the tip of the 200 l pipetman with about 10 l of the screening solution . in each case , the presence of a single fluorescent bead was confirmed by observing the pipet tip under the long wave uv lamp . the bead was transferred to a millipore ultrafree - mc spin tube by placing the bead - loaded tip of the pipetman near ( but not resting on ) the bottom of the tube before dispensing the contents . the presence of the fluorescent bead in the spin tube was confirmed under a uv lamp using a stereo microscope . one , the prewashing of the millipore ultrafree - mc spin tube consisted of a water wash ( 2  250 l ) , followed by a methanol wash ( 2  250 l ) , and finished with a dichloromethane wash ( 2  250 l ) . two , just before adding the cnbr solution , the beads are placed in a drying oven for 5 min , then removed and allowed to cool for an additional 5 min .