text: sinapinic acid ( dimethoxy hydroxycinnamic acid ) ( aldrich chemical co ., inc ., milwaukee , wis .) 20 mg / ml water suspension ( intrinsic ph 3 . 88 ) cyanohydroxycinnamic acid ( aldrich ) 20 mg / ml water suspension ( intrinsic ph 3 . 3 ) each titrated with triethylamine ( pierce , rockford , ill .) to ph 6 . 5 , 7 . 2 and 6 . 5 respectively 2 ul of the matrix solution was mixed with 1 ul of sample and allowed to air dry 1 . a mixture of synthetic peptides — human bistidine - rich glycoprotein peptide ( ghhph ) 2 g , ( ghhph ) 5 g , human estrogen receptor dimerization domain ( d473 - l525 ) with neutralized sinapinic acid as the matrix , in the absence and presence of cu ( ii ). fig1 showed the in situ metal - binding properties of the peptides under neutralized condition . 2 . casein phosphopeptide ( r1 - k18 + 5p ) with sinapinic acid ph 6 . 5 . followed by in situ alkaline phosphatase ( 0 . 5 ul , sigma ) digestion for 10 min at room temperature . similar in situ digestion on the same peptide with dihydoxybenzoic acid ( prepared in 30 % methanol / 0 . 1 % trifluoroacetic acid ) was used as control . fig2 showed the more efficient enzymatic dephosphorylation under neutralized condition . 3 . mixture of synthetic peptides as in 1 with sinapinic acid ph 6 . 2 , followed by in situ carboxypeptidase p ( 1 ul , boehringer mannheim corp , indianapolis , ind ., 20 ug / 50 ul ) digestion for 30 min at room temperature . fig3 showed preferential removal of c - terminal amino acid from histidine - rich glycoprotein peptide . also showed unambiguous c - terminal determination even in peptide mixtures . usage of probe tip ( surface ) materials ( composition ) other than stainless steel or platinum for sample deposition molten polypropylene or polystyrene was deposited on stainless steel probe tip so as to cover it completely solid glass rod ( 1 . 5 mm dia ) was cut into 1 cm segments and inserted into stainless steel probe tip support solid nylon ( trimmer line , 1 . 5 mm dia , arnold , shelby , ohio ) was cut into 1 cm segments and inserted into stainless steel probe tip support magnetic stir bars ( 1 . 5 × 8 mm , teflon coated , curtin matheson scientific , inc ., houston tex .) inserted into stainless steel probe tip support peptide mixtures ( as in fig1 with dihydroxybenzoic acid in 30 % methanol / 0 . 1 % tfa ) on all four surfaces . fig4 affinity - directed laser desorption ( with matrix added as described in prior art ) cu ( ii ) was chelated by iminodiacetate group covalently attached to either porous agarose beads ( chelating sepharose fast flow , pharmacia biotech inc ., piscataway , n . j ., ligand density 22 - 30 umole / ml gel ) or solid tsk - sw beads ( toyosoda , japan , ligand density 20 umole / ml gel ) fe ( iii ) was chelated by tris ( carboxymethyl ) ethylenediamine - sepharose 6b ( synthesized as described by yip and hutchens , protein expression and purification 2 ( 1991 ) 355 - 362 , ligand density 65 umole / ml ) 1 . a mixture of synthetic peptides , neurotensin ( 30 nmole ), sperm activating peptide ( 50 nmole ) and angiotensin i ( 150 nmole ), were mixed with 50 ul packed volume of tsk sw - ida - cu ( ii ) at ph 7 . 0 ( 20 mm sodium phosphate , 0 . 5 m nacl ) at room temperature for 10 min . the gel was then washed with 3 × 200 ul sodium phosphate buffer , containing 0 . 5 m nacl , ph 7 . 0 and suspended in equal volume of water . 2 ul of the gel suspension was mixed with 1 ul sinapinic acid ( methanol ). fig5 a showed the molecular ions ( and multiple na - adducts ) of neurotensin and sperm activating factor which were not adsorbed by the ida - cu ( ii ). the mass spectrum in fig5 b showed mainly the angiotensin i plus na - adducts . when the ida - cu ( ii ) gel was further washed with 500 ul of water 2 ×, the resulting mass spectrum showed only the parent angiotensin i species with no other adduct peaks . when the ida - cu ( ii ) gel beads with adsorbed angiotensin was incubated with cyanohydroxycinnamic acid ( 20 mg / ml water ) ph 7 . 0 for 10 min at room temperature and then analyzed separately , the angiotensin i was found to be still associated with the gel beads and not with the matrix solution . 2 . horse heart myoglobin ( 325 pmole ) was mixed with 50 ul of tsk sw - ida - cu ( ii ) gel beads in 20 mm sodium phosphate , 0 . 5 m nacl , ph 7 . 0 at room temperature for 10 min . the gel beads were then washed with 2 × 500 ul of buffer and 2 × 500 ul of water . the beads were suspended in equal volume of water and then serial diluted into water . 0 . 5 ul of the diluted gel suspension was mixed with 1 ul of sinapinic acid ( 30 % methanol / 0 . 1 % tfa ). a detectable signal ( after averaging 50 laser shots ) of myoglobin was still obtained when the calculated quantity equivalent to or less than 8 fmole was deposited on the probe tip . fig6 . 100 ul of infant formula and gastric content of preterm infant aspirated 90 min after feeding of infant formula was mixed with 50 ul of ted - fe ( iii ) in 0 . 1 m mes , 0 . 15 m nacl , ph 6 . 5 at room temperature for 15 min . the gel beads were then washed with 3 × 500 ul of mes buffer and then with 1 × 500 ul of water . 1 ul of the gel suspension was mixed with 2 ul of sinapinic acid ( 50 % acetonitrile / 0 . 1 % tfa ). the result showed that gastric aspirate had much more low molecular weight phosphopeptides ( i . e ., bound by ted - fe ( iii )) than the formula due to proteolytic digestion . in situ alkaline phosphatase digestion of peptides adsorbed on the ted - fe ( iii ) gel beads showed shifts to lower molecular weight indicating that they are indeed phosphopeptides . fig7 polyclonal rabbit anti - human lactoferrin antibody was custom generated for this lab by bethyl laboratories ( montgomery , tex .). it was purified by thiophilic adsorption and then by immobilized lactoferrin column . sheep anti - rabbit igg covalently attached to magnetic beads were obtained from dynal as ( oslo , norway ) ( uniform 2 . 8 um superparamagnetic polystyrene beads , ligand density 10 ug sheep igg per mg bead ). 1 . human lactoferrin ( 1 nmole ) was incubated with rabbit antihuman lactoferrin at 37 ° for 30 min . subsequently , 40 ul of sheep anti - rabbit igg on dynabeads ( 6 - 7 × 10 8 beads / ml ) was added and incubated at 37 ° for 30 min . the beads were then washed with 3 × 500 ul of sodium phosphate buffer , and 2 × 500 ul of water . the final amount of human lactoferrin bound to the complex was estimated to be 4 pmole . approximately 1 / 10 of the beads was transferred to a magnetic probe tip and mixed with 2 ul of sinapinic acid ( 30 % meoh / 0 . 1 % tfa ). result showed the lactoferrin ion signal in addition to the rabbit igg signal . fig8 group 3 . immobilized nucleic acid as the affinity ligand single - strand dna immobilized on 4 % agarose beads was obtained from gibco brl , gaithersburg , md . the ligand density was 0 . 5 - 1 . 0 mg / ml . 1 . 200 ul of 125 i human lactoferrin ( equivalent to 49 nmole ) was mixed with 100 ul of immobilized single - strand dna in 20 mm hepes , ph 7 . 0 at room temperature for 10 min . the beads were then washed with 5 × 500 ul of hepes buffer and then suspended in equal volume of water . the amount of lactoferrin bound per bead was found to be 62 fmole by determining the radioactivity and counting the number of beads per unit volume . various numbers of beads ( from 1 to 12 ) were deposited on 0 . 5 mm diameter probe tips and mixed with 0 . 2 ul of sinapinic acid ( 30 % methanol / 0 . 1 % tfa ). lactoferrin ion signals were obtained with multiple 100 laser shots on a single bead with adsorbed lactoferrin . fig9 . 30 pmole of 59 fe - human lactoferrin was added to 1 ml of preterm infant urine and mixed with 20 ul of immobilized single - strand dna on agarose in 0 . 1 m hepes ph 7 . 4 at room temperature for 15 min . the beads were washed with 2 × 500 ul hepes buffer , and 2 × 500 ul of water . the beads were then suspended in equal volume of water and 1 ul ( equivalent to not more than 350 fmole as determined by radioactivity ) was mixed with 1 ul sinapinic acid ( 30 % methanol / 0 . 1 % tfa ) on a probe tip . positive lactoferrin signals were obtained for multiple 50 laser shots . fig1 group 4 . immobilized biomolecule as the affinity ligand soybean trypsin inhibitor ( sigma , st louis , mo .) was immobilized on affigel 10 ( biorad laboratories , hercules , calif ., ligand density 15 umole / ml ) according to manufacturer &# 39 ; s instructions . 1 . 100 ul of human gastrointestinal aspirate was mixed with 50 ul of immobilized soybean trypsin inhibitor in 20 mm sodium phosphate , 0 . 5 m sodium chloride , ph 7 , at room temperature for 15 min . the gel beads were then washed with 3 × 500 ul of phosphate buffer , and 2 × 500 ul of water . 1 ul of gel bead suspension was mixed with 2 ul of sinapinic acid ( 50 % acetonitrile / 0 . 1 % tfa ). result showed the presence of trypsin and trypsinogen in the aspirate . fig1 cibacron blue 3ga - agarose ( type 3000 , 4 % beaded agarose , ligand density 2 - 5 umoles / ml , sigma ). other immobilized dyes include reactive red 120 - agarose , reactive blue - agarose , reactive green - agarose , reactive yellow - agarose ( sigma ) 1 . 200 ul of human plasma was mixed with 50 ul of immobilized dye in 20 mm sodium phosphate , 0 . 5 m nacl , ph 7 . 0 at room temperature for 10 min . the gel beads were then washed with 3 × 500 ul of phosphate buffer and 2 × 500 ul of water . 1 ul of gel bead suspension was mixed with 2 ul of sinapinic acid ( 50 % acetonitrile / 0 . 1 % tfa ). result showed the selective adsorption of human serum albumin from the serum sample by cibacron blue . fig1 group 1 . energy - absorbing molecule covalently bonded to surface via the n - group cinnamamide ( aldrich , not a matrix by prior art ) was dissolved in isopropanol / 0 . 5 m sodium carbonate ( 3 : 1 ) and mixed with divinyl sulfone ( fluka , ronkonkoma , n . y .) activated sepharose ( pharmacia ) at room temperature for 2 hr . the excess molecules were washed away with isopropanol . the proposed structure was presented in fig1 . 2 ul of bound or free molecule was deposited on the probe tips , 1 ul of peptide mixtures in 0 . 1 % tfa was added on top and the result showed the peptide ion signals detected only for the bound form . fig1 . group 2 . energy absorbing molecule covalently bonded to surface via the c - group cynnamyl bromide ( aldrich , not a matrix by prior art ) was dissolved in isopropanol / 0 . 5 m sodium carbonate and mixed with divinyl sulfone - activated sepharose at room temperature overnight . the excess molecules were washed away with isopropanol . the proposed structures are presented in fig1 . 2 ul of the bound or free molecule was deposited on the probe tip , 1 ul of peptide mixtures in 0 . 1 % tfa was added on top and the result showed the detection of peptide ion signal only for the bound form . fig1 . group 3 . energy absorbing molecule covalently bonded to surface via the c - group dihydroxybenzoic acid was activated by carbodiimide and mixed with fmoc - map 8 branch resin ( applied biosystems , forster city , calif .) overnight . the proposed structure was presented in fig1 . after washing , 1 ul of the bonded molecule on map or the map alone in 50 % acetonitrile / 0 . 1 % tfa were deposited on the probe tip , 1 ul of peptide mixture was added on top , the resulting mass spectrum was presented in fig2 . group 4 . energy absorbing molecule covalently bonded to surface via undetermined group . cyanohydroxycinnamic acid was dissolved in methanol and mixed with affigel 10 ( biorad ) at room temperature for two hours . the unbound molecules were washed away with methanol . protein samples that are found to desorb successfully from this modified surface include myoglobin ( fig1 ), trypsin and carbonic anhydrase . these examples ( groups 1 - 4 ) are also demonstrations of combined surface - enhanced and affinity - directed desorption where the adsorbed ( bonded ) energy absorbing molecular also act as affinity adsorption reagents to enhance the capture of analyte molecules . ( 1 ) “ presenting surface ”— the probe tip , sample plate or other surface on which the analyte and matrix are presented for desorption / ionization and analysis for example by mass spectrometry . ( 2 ) “ matrix ”— as described in prior art as the substance mixed with the analyte ( typically prior to deposition ) and deposited on the presenting surface in association with the analyte to absorb at least part of the energy from the energy source ( e . g ., laser ) to facilitate desorption of intact molecules of the analyte . ( 3 ) “ analyte ”— the material which is the subject of desorption and investigation by mass spectrometry or other means for detection . ( 4 ) “ affinity reagent ” ( analyte capture device )— the class of molecules ( both man made , unnatural , natural and biological ) and / or compounds which have the ability of being retained on the presenting surface ( by covalent bonding , chemical absorption , etc .) while retaining the ability of recognition and bonding to an analyte . ( 5 ) “ desorption ”— the departure of analyte from the surface and / or the entry of the analyte into a gaseous phase . ( 6 ) “ ionization ”— the process of creating or retaining on an analyte an electrical charge equal to plus or minus one or more electron units . ( 7 ) “ adduct ”— the appearance of an additional mass associated with the analyte and usually caused by the reaction of excess matrix ( or matrix break - down products ) directly with the analyte . ( 8 ) “ adsorption ”— the chemical bonding ( covalent and / or noncovalent ) of the energy - absorbing molecules , the affinity reagent ( i . e ., analyte capture device ), and / or the analyte to the probe ( presenting surface ).