Matrix-assisted laser desorption/ionization. Statistical Evaluation of Electrospray Tandem Mass Spectra for Optimized Peptide Fragmentation

Transcription

1 Sttistil Evlution of Eletrospry Tnem Mss Spetr for Optimize Peptie Frgmenttion Json C. Roglski, Mihel S. Lin, Mtthew J. Snitynski, n Roert J. Tylor The Biomeil Reserh Center, University of British Columi, Vnouver, British Columi, Cn Nikoly Youhnovski n Mihel Przyylski Deprtment of Chemistry, University of Konstnz, Konstnz, Germny Juergen Kst* Deprtment of Chemistry, University of British Columi, Vnouver, British Columi, Cn Liqui hromtogrphy-tnem mss spetrometry (LC-MS/MS) hs eome the metho of hoie for the nlysis of omplex peptie mixtures. It omines the seprtion power of nnoflow LC with highly speifi sequene nlysis, llowing utomte peptie sequening with high resolution n throughput. For peptie frgmenttion, the urrent experimentl setup uses preefine prmeters se on the mss-to-hrge rtio of the iniviul preursor. Suitle prmeters re typilly estlishe y empiril evlution of frgment spetr of iniviul pepties use s stnrs. As result, nonoptiml frgment spetr re otine if pepties show frgmenttion ehvior ifferent from these stnrs, whih often result in the loss of sequene-speifi frgment ion informtion. Here we esrie sttistil pproh for the systemti evlution of the qulity of iniviul peptie frgment spetr se on the lultion of their rithmeti men n stnr evition. The metho utilizes the epenene of these prmeters on the ifferene in eletri potentil ross the ollision ell to etermine the vlue tht results in mximum informtion ontent. We show tht the metho is pplile to frgment spetr generte from vriety of multiply-hrge trypti pepties, over wie onentrtion rnge, n on ifferent types of mss nlyzers. We lso show how this novel pproh n e use to efine optimize ollision energy settings over wie mss-to-hrge rnge. (J Am So Mss Spetrom 25, 16, ) 25 Amerin Soiety for Mss Spetrometry Mtrix-ssiste lser esorption/ioniztion (MALDI) n eletrospry ioniztion (ESI) se mss spetrometry hs evelope into n inispensle tool for reserhers in moleulr iology n iotehnology, n key tehnology in proteomis [1]. Two ifferent pprohes inorporting mss spetrometry hve emerge for the ientifition of proteins fter enzymti igestion into pepties. Peptie mss fingerprinting etermines the msses of ll pepties present in the igestion mixture n ompres the resulting ptterns to those preite for ll entries in protein or genome sequene tses. This pproh usully employs MALDI-MS euse of its inherent simpliity, spee, n reliility, ut is limite Pulishe online Ferury 1, 25 Aress reprint requests to Dr. J. Kst, The Biomeil Reserh Center, University of British Columi, 2222 Helth Sienes Mll, Vnouver, BC V6T 1Z3, Cn. E-mil: kst@r.u. to igests of isolte proteins or simple protein mixtures [2]. For more omplex igestion mixtures, peptie sequening using tnem mss spetrometry omine with liqui hromtogrphy (LC-MS/MS) is pplie [3]. In this pproh, iniviul pepties re seprte, isolte from the mixture, n frgmente using low or high-energy ollisions. In orer to ientify the est-mthing sequene, the frgmenttion ptterns otine for speifi peptie re ompre to those ntiipte for eh hypothetil peptie preite from ll tse entries. Although reently introue MALDI-MS/MS tehniques now lso permit seletion n sequening of iniviul pepties [4, 5], the vst mjority of peptie sequening experiments re still rrie out y ESI-MS/ MS. Prtiulrly, the genertion of intense frgment ions from multiply-hrge peptie ions n the possiility of on-line seprtion y multi-imensionl hromtogrphy mke ESI-MS/MS vlule for peptie sequening [6]. Consequently, severl high-throughput tehniques suh 25 Amerin Soiety for Mss Spetrometry. Pulishe y Elsevier In. Reeive Ferury 18, /5/$3. Revise Jnury 4, 25 oi:1.116/j.jsms Aepte Jnury 4, 25

2 56 ROGALSKI ET AL. J Am So Mss Spetrom 25, 16, s isotope-oe ffinity tgging (ICAT) [7] n multiimensionl protein ientifition tehnology (MuPit) [8] rely on this pproh to sequene thousns of pepties in one experiment. The suessful ientifition of proteins y peptie sequening is epenent on three onitions: (1) the existene, orretness, n uniqueness of the mthing tse entry, (2) the ientifition n seletion of the preursor peptie ions for frgmenttion, n (3) the genertion of suffiient tnem mss spetrometry informtion. Only if ll of them re met re proteins ientifie with high onfiene. Consequently, severl pprohes hve een evelope to ientify proteins tht re not present in tses [9 14] n to inrese the sensitivity for eteting preursor ions [15 17]; however, the qulity of frgment spetr hs rrely een resse. Although methos to exlue frgment spetr of insuffiient qulity prior to tse serhing [18] n to evlute the resulting sores [19 23] hve een reporte, pprohes tht llow systemti optimiztion of quisition prmeters suh s the eletri potentil tht inues peptie frgmenttion (ollision energy) re lking. Inste, they re usully estlishe empirilly y frgmenting stnr pepties t vrious ollision energies, seleting the frgmenttion spetrum tht ppers to ontin to most sequene informtion, n trnsferring the orresponing ollision energy n mss-to-hrge (m/z) rtio of the stnr peptie into referene tle. In n LC-MS/MS experiment, instrument softwre then etermines the m/z vlue of n eluting peptie n sets the ollision energy for frgmenttion oring to this tle. This n result in nonoptiml frgment spetr if peptie shows frgmenttion ehvior ifferent from tht of the stnr pepties, whih woul use loss of sequene-speifi frgment ions n ompromise sequene informtion. In more reent pprohes, rnge of eletri potentils is therefore pplie y stepping or rolling the ollision energy roun the preefine vlue, whih inreses the likelihoo of rnomly generting suffiient peptie frgment ions [13, 24]. Due to the lk of sttistil t, the tul size of the rnge is still etermine empirilly. Here we esrie n lterntive pproh se on the reltionship etween the ollision energy n the informtion ontent of peptie frgment spetr. We mke use of the rithmeti men n the stnr evition s two prmeters tht n e use to etermine the mein n spre of t istriutions. By systemtilly ssessing the epenene of these prmeters on the potentil ifferene etween the qurupole ion guie (Q) n the ollision ell (Q2), we show tht they re suffiient to esrie the qulity of frgmenttion spetr. These lultions re not signifintly ffete y the peptie type, its sequene, its hrge stte, or the genertion of unnt low-mss frgment ions. Furthermore, we show tht our metho is omptile with rnge of peptie onentrtions n instrument types. The metho reily lens itself to the optimiztion of ollision energy settings over wie mss-to-hrge rnge. Experimentl Chemils Brykinin frgment 1-9 (RPPGFSPFR, verge MW D), Brykinin frgment 2-9 (PPGFSPFR, verge MW 94.3 D), ovine serum lumin (BSA), -sein, fetuin (ll ovine), n iooetmie were purhse from Sigm-Alrih (St. Louis, MO), sequening gre trypsin ws otine from Promeg (Mison, WI), n ithiothreitol ws from Amershm (Pistwy, NJ). All solvents use were of HPLC gre. For trypti igests, proteins were enture t 9 C for 3 min, reue with ithiothreitol, lkylte with iooetmie, n igeste in solution using enzyme/sustrte rtio of 1:5 (wt/wt) overnight s esrie [25]. Protein igests n iniviul pepties were issolve in methnol/wter/formi i (5: 45:5) to finl onentrtions of 2 pmol/ l (igests) n 2 fmol/ l, 2 fmol/ l, n 2 pmol/ l (pepties), prior to mss spetrometri nlysis. Mss Spetrometry For t quisition, 1 5 l of the vrious peptie solutions were trnsferre into iniviul nnoeletrospry neeles (Protn, Oense, Demrk). Nnoeletrospry mss spetometry experiments of iniviul pepties were performe on hyri qurupole-time of flight instrument (Q-Str Pulsr i, Applie Biosystems, Frminghm, MA) supplemente y ommeril nnoeletrospry interfe (Protn). In ition, triple qurupole instrument (API III, Siex, Conor, ON, Cn) equippe with ustom-uilt nnoeletrospry interfe, n Fourier Trnsform-Ion Cylotron Resonne (FT-ICR) instrument (APEX II, Bruker Dltonik, Bremen, Germny) with n tively shiele 7. tesl superonuting mgnet (Mgnex, Oxfor, UK) n uilt-in nnoeletrospry interfe (APOLLO, Bruker Dltonik) were use. A seon hyri qurupole - time of flight instrument (Q-Str Pulsr XL, Applie Biosystems) ws employe for the nlysis of protein igests n the lirtion plot. Tnem mss spetr quire on qurupole-time of flight instruments were verge for t lest one minute, wheres t lest five sns were verge on the triple qurupole instrument, epening on the peptie onentrtion. FT-ICR tnem mss spetr were otine y olleting 16 single sns using experimentl onitions s esrie [26]. For the lirtion plot, potentil ifferenes were steppe y 2 V with n quisition time of 15 s per inrement for eh of the 22 preursor ions. Aquisition of ll spetr ws performe with the respetive instrument softwre. Dt Proessing All tnem mss spetr were otine in uplite (lirtion plot) or triplite (ll other experiments)

3 J Am So Mss Spetrom 25, 16, STATISTICAL EVALUATION OF PEPTIDE TANDEM MASS SPECTRA 57 n onverte into ASCII formt, proesse with inhouse softwre, n importe into Exel (Mirosoft, Mississug/ON, Cn) for t isply. The inhouse softwre ws written in C n use the following equtions: : 1 (x i y i ) (1) N i 1 : 2 1 N 1 2 (x i ) (2) N i 1 with: x iniviul mss-to-hrge rtio, y intensity t x, N totl numer of mss-to-hrge rtios, to mthemtilly esrie the weighte verge (rithmeti men ) n the spre (stnr evition ) of t istriution. For etermintion of frgment ion overge, spetr were entroie in Anlyst QS (Applie Biosystems, Foster City, CA) using 6 ppm merge istne, 99% height, n 1 n 5 ppm mimimum n mximum pek withs, respetively, n onverte into ASCII formt. Theoretil msses of ll - n y-type s well s immonium n internl frgment ions were ompre to eh of these spetr n ounte if they fell within.1 D mss error. Frgment ion types were ssigne oring to the nomenlture of Roepstorff n Fohlmn [27] moifie y Biemnn [28]. For noise filtering of FT-ICR t, isrete wvelet enoising opertion [29] ws performe using 1- point Duehies wvelet n hr threshol funtion tht zeroe the lowest 99.9% of points in the wvelet trnsforme t. This remove muh of the noise, leving signl peks isernily ove the remining seline. The remining seline ws remove y sutrting 1.5 times the verge of lol mxim in 1,1-point winow roun eh t point. Results n Disussion Frgment Ion Coverge In peptie sequening, the most importnt riterion for suessful protein ientifition is the genertion of sequene-speifi frgment ions, whih iretly reltes to the sore hieve in tse serh [3]. The totl numer of mthing frgment ions is epenent on severl experimentl prmeters, inluing the iniviul peptie sequene n the ollision energy settings. The ollision energy, whih reflets the kineti energies of preursor ions in the ollision ell, n e etermine y multiplying the hrge stte of the preursor ion with the potentil ifferene etween the qurupole ion guie (Q) n the ollision ell (Q2). We wishe to gin insight into how vrious potentil ifferenes ffet the totl numer of mthing frgment ions for pepties with similr msses, ut ifferent sequenes. For three ouly-hrge peptie ions generte from trypti igests of -sein n ovine serum N lumin, we etermine the numer of mthing frgment ions for potentil ifferenes inrese from 15 to 5 V in 5 V inrements (Figure 1). The frgment ions were groupe into those proue y frgmenttion of single peptie on (- n y-ions, positive sle) or two peptie ons (immonium n internl frgment ions, negtive sle), respetively [31]. Frgment ions generte y itionl sie hin frgmenttion, suh s y-nh 3, -NH 3, -H 2 O, or y oth one- n two-step frgmenttion pthwys, i.e., -type ions, oul not e ssigne to either group n were thus not onsiere. The resulting plots show similr trens with inresing ollision energy for ll pepties, nmely n initil inrese, mximum, n susequent erese in the numer of mthing frgments. Closer inspetion inites tht the potentil ifferene t whih the mximum numer of mthing frgment ions is otine, vries from peptie to peptie. For -sein (FFVAPFPEVFGK) oserve t m/z , mximum of 19 mthing - n y-ion frgments is rehe t 25 n 3 V (positive sle), while the mximum numer of 37 mthing immonium n internl frgment ions re oserve t 4 V (negtive sle). Similr results re foun for BSA (TVMENFVAFVDK) etete t m/z 7.35 with mximum of 19 mthing - n y-type ions t 25 V n 29 mthing immonium n internl frgment ions t 4 V. By ontrst, for BSA 89-1 (SLHTLFGDELCK) ientifie t m/z 71.35, mximum of 19 mthing -n y-type n 34 mthing immonium n internl frgment ions re oserve t 4 n 5 V, respetively. Frgmenting this peptie t the potentil ifferenes tht generte the mximum Numer of Mthes Figure 1. Depenene of the numer of mthing frgment ions on the potentil ifferene use for peptie frgmenttion for the ouly-hrge trypti pepties -sein t m/z , (lk), BSA t m/z 7.35 (grey), n BSA 89-1 t m/z (white) on hyri qurupole-time of flight instrument. The numers of mthing - n y-ions re epite on the positive sle, mthing immonium n internl frgment ions on the negtive sle.

4 58 ROGALSKI ET AL. J Am So Mss Spetrom 25, 16, numer of mthing frgment ions for the forementione two pepties, i.e., 25 n 4 V, results in 7 fewer - n y-type n 11 fewer immonium n internl frgment ions. Consequently, pplying onitions tht re se on the frgmenttion of pepties with similr m/z rtio orrespons to signifint loss of peptiespeifi frgment ions. Moreover, for ll three pepties, reking two peptie ons requires 1 15 V higher potentil ifferenes thn reking single peptie ons. Although this is esily expline y the inrese in intrinsi energy require for this proess, the prtil onsequenes for peptie sequening re still noteworthy. Dtse serh progrms im to orrelte ll experimentlly oserve frgment ions with those tht re generte in silio for eh sequene se on preefine frgment ion types [3], therefore the iel potentil ifferene for eh peptie shoul oinie with the highest totl numer of mthing frgment ions. We foun the numer of mthing immonium n internl frgment ions to e signifintly higher thn the - n y-type frgment ions in ll ses, therefore the sum of oth is ise towrs frgment ions tht ontin less sequene informtion. On the other hn, onsiering only - n y-type frgment ions woul isr vlule informtion tht n e use to vlite the peptie sequene. Our t inite tht mximum sequene onfiene is hieve t potentil ifferenes slightly higher thn inite y the - n y-type frgment ion mximum, euse shrp inrese in the numer of mthing immonium n internl frgment ions ompenstes the slight erese in the numer of mthing - n y-type frgment ions. In our stuy, using three pepties with lmost ientil moleulr weight n hrge stte, we foun tht the potentil ifferene require to hieve optiml frgmenttion n iffer y more thn 1 V. We onlue tht the mss-to-hrge rtio lone is not goo initor to etermine the optiml potentil ifferene, n tking into ount the iniviul frgment spetr might e more suite for this purpose. Evlution of Iniviul Frgment Ion Distriutions In orer to investigte wht the hrteristis of the iniviul spetr re n how they hnge with inresing potentil ifferene, series of tnem mss spetr using moel peptie mimiking the sequene of trypti peptie (Brykinin frgment 2-9, sequene PPGFSPFR) were generte. Its oulyhrge moleulr ion t m/z ws selete n frgmente on hyri qurupole-time of flight instrument ltering the potentil ifferene from 1 to 5 V in 5 V inrements. At 2 V potentil ifferene (Figure 2), mostly the preursor ion t m/z is visile. Uner these onitions, the initil threshol of frgmenttion is 1% % 1% % m/z m/z e % rely overome, leving the preursor ion lrgely intt while reking only the most lile ons. Due to the nture of the trypti peptie, the si roxyterminl mino i resiue will retin one proton reting frgment ion of the y-series while itionl protons re moile n istriute over the entire peptie [32]. A neutrl mino-terminl frgment will only e rete if the on rekge ours lose to the mino terminus of the peptie, otherwise two singlyhrge frgments with omplementry msses will e otine. A ouly-hrge frgment ion t m/z (y7 ) with signifint intensity is ue to the loss of the mino-terminl proline resiue, whih proves the proline-proline on to e the most lile [31]. A numer of singly-hrge frgment ions of the y series re present in the high-mss prt of the spetrum ut show very low intensity. Rising the potentil ifferene to 25 V (Figure 2) signifintly inreses the intensities of the previously oserve frgment ions t m/z (y4), (y5), (y6), n (y7), % 1% % m/z m/z Figure 2. Representtive frgment spetr of ouly-hrge Brykinin 2-9 (m/z ) otine on hyri qurupole-time of flight instrument t () 2V,() 25V,() 35 V, n () 45V potentil ifferene. Frgment spetr were quire in triplite, inresing the potentil ifferene in 5 V inrements, rithmeti men (e) n stnr evition (f) were lulte oring to eqs 1 n 2 n plotte ginst the potentil ifferene. f

5 J Am So Mss Spetrom 25, 16, STATISTICAL EVALUATION OF PEPTIDE TANDEM MASS SPECTRA 59 n yiels itionl ions in the low-mss en of the spetrum. These frgment ions re generte with similr proilities n hve similr intensities euse the frgmenting ons hve omprle stilities. An intense signl is still oserve for the nonfrgmente preursor ion, initing tht the kineti energy, whih will follow Mxwell-Boltzmnn istriution, is insuffiient to inue frgmenttion for signifint portion of the preursor ion. By ontrst, t 35 V (Figure 2), the preursor ion is ompletely sent n the singly-hrge frgment ion y3 with n itionl loss of mmoni (y3-nh 3 )tm/z eomes the se pek of the spetrum. In ition to the frgment ions oserve t 25 V, there is lrge numer of low-mss ions. These re immonium ions suh s those of proline (m/z 7.7) n phenyllnine (m/z 12.8), terminl frgment ions of the -, -, n y-series suh s those t m/z (y1) n 98.6 (1), n internl frgment ions suh s those of PG (m/z 155.8) n PF with n itionl loss of ron monoxie CO (m/z ). These ions re forme y seonry frgmenttion of the lrge peptie frgments. Therefore, onurrent erese of lrge frgment ions n e notie for the inrese in intensity of the low-mss frgment ions. At 45 V (Figure 2), the low-mss frgment ions ominte the spetrum while high-mss frgment ions re virtully sent, initing seonry frgmenttion is ommonple. In orer to etermine whether the vrying istriution of frgment ions oinies with hnge in the totl numer of sequene-speifi frgment ions, we rete pek list for eh of these frgment spetr n ompre them to the list of ll possile -, y-, internl, n immonium ions we preite for Brykinin frgment 2-9, using the previously esrie riteri. At 2 V, only one out of mximum of 35 possile frgment ions is foun, wheres 11 possile frgment ions re oserve t 25 V. The numer of mthing frgment ions rehes mximum of 26 t 35 V potentil ifferene efore elining to 17 t 45 V. From these experiments, we onlue tht onitions tht result in the spetr shown in Figure 2,, n likely o not generte enough sequene-speifi informtion for unmiguous ientifition. By ontrst, onitions tht le to the spetrum in Figure 2 proue the highest numer of sequene-speifi peptie frgments n therefore orrespon to the highest likelihoo of unmiguous ientifition. n We foun wie istriution of frgment ions to e key feture of high-soring frgment spetr. Therefore, we teste whether the rithmeti men, mthemtil vrile tht esries the weighte verge of t istriution (eq 1), n the stnr evition, whih is use to quntify the spre of suh t istriutions (eq 2), oul e pplie for the evlution of frgment spetr. For the series of spetr otine in the previously esrie experiment, the rithmeti men n stnr evition were lulte n plotte ginst the iniviul potentil ifferene settings. The rithmeti men (Figure 2e) remins lmost onstnt for potentil ifferenes from 15 to 3 V with vlue similr to the mss-to-hrge rtio of the preursor ion, followe y shrp rop from 35 to 5 V. This inites tht the entre of the frgment ion istriution eomes progressively lower thn the preursor m/z ue to the inresing presene of low-mss frgments. By ontrst, the stnr evition (Figure 2f) is low t 1 V ue to only few frgment ions eing present. It ontinuously inreses from 15 to 3 V where it rehes mximum, initive of the wiest spre of informtion in the spetrum, then rops off t 35 to 5 V. From these results, we onlue tht the frgment spetrum possessing the most even istriution of frgment ions, epite in Figure 2, will show n rithmeti men similr to the preursor m/z, n stnr evition tht is mximl. Therefore, lulting rithmeti men n stnr evition is suffiient to istinguish potentil high- n low-soring frgment spetr. Evlution of Mxim The previous t inite tht the optiml potentil ifferene is etween 25 n 35 V. Therefore, we quire seon set of frgment spetr using the sme onitions ut ltering the potentil ifferene from 25 to 35 V in 1 V inrements. By ompring the spetr t 25 V (Figure 2) n 35 V (Figure 2), itis notiele tht the mjor ifferene etween these spetr is in the reltive intensity of the vrious frgment ions rther thn their mere presene. This tren is onfirme in representtive frgment spetr shown for inresing potentil ifferene. At 27 V (Figure 3), the se pek of the spetrum is the ouly-hrge frgment ion t m/z (y7 ) with the preursor ion t m/z hving omprle intensity. While high-mss frgment ions t m/z (y4), (y5), (y6), n (y7) show signifint intensities, low-mss frgment ions t m/z 7.7 (immonium ion, proline), m/z (internl frgment PG), n m/z (y1) show lower intensities. When the potentil ifferene is rise to 29 V (Figure 3), the intensity of the ouly-hrge frgment ion t m/z (y7 ) ereses while high- n lowmss frgment ions gin in intensity, with the one t (y6) eoming the se pek. At 31 V (Figure 3), the low-mss frgment ion t m/z 7.7 (immonium ion of proline) eomes the se pek, with intense lown high-mss frgment ions n the lmost omplete ispperne of the preursor ion. Finlly, when the potentil ifferene is inrese to 33 V (Figure 3), the very low-mss frgment ions gin in intensity while ll other frgment ions erese. The rithmeti men n the stnr evition were gin lulte n plot-

6 51 ROGALSKI ET AL. J Am So Mss Spetrom 25, 16, % % 7.7 % % % 7.7 m/z 1% 7.7 m/z % te for this set of frgmenttion spetr. The rithmeti men (Figure 3e) remins lmost onstnt etween 25 n 28 V, ut ereses t higher potentil ifferenes with n inresing slope. By ontrst, the stnr evition (Figure 3f) inreses n rehes mximum t 33 V efore it strts to erese. Although the hnges in the 25 to 35 V rnge seem rther sutle, omprison of the iniviul spetr inites tht they iffer signifintly in the intensities of the iniviul frgment ions. It is noteworthy tht the shift in intensities from high-mss to low-mss frgment ions is more ovious y the rop in the rithmeti men thn y the miniml hnges in the stnr evition. However, the mximum of the stnr evition t 33 V oes oinie with the spetrum tht is the most iverse in frgment ions. Chrge Stte n Peptie Sequene % m/z e Sine it is well known tht ifferent types of preursor ions generte very ifferent frgmenttion ptterns, we m/z Figure 3. Representtive frgment spetr of ouly-hrge Brykinin 2-9 (m/z ) otine on hyri qurupole-time of flight instrument t () 27V,() 29V,() 31V,n() 33V potentil ifferene. Frgment spetr were quire in triplite, inresing the potentil ifferene in 1 V inrements, rithmeti men (e) n stnr evition (f) were lulte oring to eqs 1 n 2 n plotte ginst the potentil ifferene. 3 f investigte whether the stnr evition pproh n lso e pplie to evlute frgment spetr of more omplex preursor ions tht regulrly our in trypti igests. Speifilly, we use nother moel peptie mimiking the sequene of trypti peptie ontining seon si resiue ue to misse levge site (Brykinin frgment 1-9, sequene RPPGFSPFR). For this peptie oth the ouly n triply hrge preursor ions were oserve t m/z n , respetively, s ommonly foun in trypti igests. Eh of them ws frgmente iniviully on hyri qurupole-time of flight instrument using potentil ifferene rnge from 1 to 5 V in 5 V inrements. After lulting rithmeti men n stnr evition for eh of these frgmenttion spetr, the resulting urves were plotte ginst those otine previously for Brykinin frgment 2-9. The hnge in rithmeti men with inresing potentil ifferene is remrkly similr for ll three preursor ions (Figure 4). All of them show lmost onstnt vlues until they reh threshol followe y shrp erese. The onset of the erese is notiele t 35 V for ouly-hrge Brykinin frgment 2-9 n t 3 V for ouly-hrge Brykinin frgment 1-9, while tht for triply-hrge Brykinin frgment 1-9 is t 25 V. Agin, seon set of experiments ws performe to explore these trens in more etil (Figure 4). For ouly-hrge Brykinin frgment 2-9, mximum of the rithmeti men is oserve t 28 V. This oes not our in the other two plots. Inste, plteu followe y erese is oserve for oth Figure 4. Depenene of (, ) rithmeti men n (, ) stnr evition on the potentil ifferene vrie in (, ) 5 V inrements n (, ) 2 V or 3 V inrements for: ouly-hrge Brykinin 2-9 (tringles), ouly-hrge Brykinin 1-9 (imons), n triply-hrge Brykinin 1-9 (squres) using hyri qurupole-time of flight instrument.

7 J Am So Mss Spetrom 25, 16, STATISTICAL EVALUATION OF PEPTIDE TANDEM MASS SPECTRA 511 strting t 24 V for ouly-hrge Brykinin frgment 1-9 n 18 V for triply-hrge Brykinin frgment 1-9. By ontrst, ll three pepties revel similr trens in the stnr evition plots, inluing mximum vlue for speifi potentil ifferene setting (Figure 4). This mximum is oserve t 3 V for ouly-hrge Brykinin frgment 2-9, t 35 V for ouly-hrge Brykinin frgment 1-9, n t 25 V for triply-hrge Brykinin frgment 1-9 when stepping from 1 to 5 V in 5 V inrements. Closer inspetion of the iniviul rnges using smller inrements onfirms these finings (Figure 4). For ouly-hrge Brykinin frgment 2-9, potentil ifferene of 32 V results in mximl stnr evition, wheres tht of oulyn triply-hrge Brykinin frgment 1-9 is etermine t 36 n 22 V, respetively. The similrity of these plots emonstrtes tht this metho is lso pplile for higher hrge sttes n trypti pepties ontining misse levge sites Low-Mss Region Figure 5. Depenene of (, ) rithmeti men n (, ) stnr evition on the potentil ifferene vrie in (, ) 5 V inrements n (, ) 1 V inrements for ouly-hrge Brykinin 2-9 peptie using low-mss ut-off of m/z 5 (imons), m/z 1 (irles), m/z 15 (tringles), n m/z 2 (squres) using hyri qurupole-time of flight instrument. Frgment ions of the -, -, n y-ion series s well s internl frgment ions re istriute over the entire mss rnge, wheres immonium ions representing single mino i resiues n only e foun in the low mss region. Due to this isproportionte representtion in the spetrum, immonium ions oul shift or lter the vlues of rithmeti men n stnr evition. Therefore, we eliminte inresing portions of the low mss rnge in the frgment spetr of ouly-hrge Brykinin frgment 2-9 for the vrious potentil ifferenes. The relulte rithmeti men n stnr evition were ompre to those otine for the unhnge spetr with low-mss ut-off t m/z 5. The rithmeti men of the originl spetrum shows onstnt vlues until 3 V followe y shrp rop t 35 V n higher (Figure 5). Cutting the low-mss region up to m/z 1 results in slight inrese of the rithmeti men to mximum t 3 V, while the rop t 35 V n higher is unhnge. Similr results re otine for ut-offs t m/z 15 n 2, respetively, lthough the hnges re more pronoune. Performing the sme proessing on the nrrow potentil ifferene rnge t onfirms the pperne of mximum t 3 V inste of n immeite erese t higher mss ut-offs (Figure 5). The low-mss ut-off hs n even more sutle effet on the stnr evition t vrious potentil ifferenes. For the t otine in 5 V inrements, no ifferene in the position of the mximum t 3 V n the tren of the plots is visile (Figure 5). The only notiele ifferene is the erese in the vlue of the stnr evition with inresing ut-off msses. Closer inspetion using the more nrrow inrement t onfirms grul flttening of the urves with inresing ut-off (Figure 5). Similr results were otine for triply-hrge Brykinin frgment 1-9 (t not shown). The only ifferene ws shift in the mximum of the stnr evition for the m/z 2 ut-off ompre to the others. This n e expline y the presene of itionl ouly-hrge frgment ions in this region. Our t therefore shows tht the low-mss region hs only minor influene on the rithmeti men n stnr evition of oth ouly- n triply-hrge preursor ions, emonstrting the generl ppliility of this metho. Peptie Conentrtion All t use for the vrious lultions esrie ove were otine using peptie onentrtions of 2 M. In orer to etermine whether this pproh is lso pplile t lower peptie onentrtions, we quire frgment spetr of ouly-hrge Brykinin frgment 2-9 t 2 n 2 nm onentrtions t vrious potentil ifferenes. Previous work h shown tht the swithing riteri re limiting ftor for the sensitivity of LC-MS/MS experiments, giving nnoeletrospry experiments t lest omprle sensitivity n mking them vli sustitute for sensitivity stuies [15 17]. For eh spetrum, we lulte rithmeti men n stnr evition, n ompre the resulting plots to those otine for the sme peptie t 2 M onentrtion. Using 5 V inrements (Figure 6), the rithmeti men remins nerly onstnt t low, ut shows rop t higher potentil ifferenes. For 2 M, this rop strts t 3 V, wheres t 2 nm it shifts to 25 V. At

8 512 ROGALSKI ET AL. J Am So Mss Spetrom 25, 16, nm, minor rop is lrey visile t 1 V, whih eomes more notiele t 25 V. Closer inspetion of the region form 25 to 35 V onfirms these finings (Figure 6). At 2 M onentrtion, mximum is rehe t 28 V efore the rithmeti men strts to erese, wheres t 2 nm there is not isernle mximum, ut erese is otine eginning t 27V. By ontrst, t 2 nm erese is oserve over the entire rnge, whih eomes more pronoune etween 25 n 3 V. Sine the vlues of the rithmeti men iffer y only 15% in this region, it eomes iffiult to etermine tren. Similr results re otine for the plots of the stnr evition t vrying potentil ifferenes. The ler mximum oserve t 3 V using peptie onentrtions of 2 M is lso oserve t 2 n 2 nm, lthough the tren is less notiele ue to inrese flttening of the urves (Figure 6). Closer inspetion of the 3 V region shows tht the mximum is shifting towrs lower potentil ifferenes with eresing onentrtions. At 2 M, the mximum is oserve t 32 V, wheres t 2 n 2 nm it shifts to 3 V. Agin, it is iffiult to lote the mximum in the plot of 2 nm euse the iniviul vlues hnge y only 15%. By ompring the iniviul spetr, we onlue tht the flttening of the urves is ue to rsti hnge of the signl-to-noise rtio use y the erese in peptie signl n the onurrent inrese in hemil noise. In ft, this is well-known effet in nnoeletrospry n LC-MS experiments ue to the genertion of lrge numers of roplets tht o not ontin ny peptie moleules, resulting in inrese Figure 6. Depenene of (, ) rithmeti men n (, ) stnr evition on the potentil ifferene vrie in (, ) 5 V inrements n (, ) 1 V inrements for ouly-hrge Brykinin 2-9 peptie using onentrtions of 2 M (imons), 2 nm (squres), n 2 nm (tringles) using hyri qurupole-time of flight instrument. 6 3 intensities of hrge solvent lusters [33]. Applition of estlishe methos to inrese signl-to-noise rtios my e use to further exten the sensitivity of this pproh [16]. Instrument Type Next, we wnte to etermine whether our metho is pltform-inepenent n omptile with ifferent types of mss nlyzers. To this en, we quire frgment spetr of ouly-hrge Brykinin frgment 2-9 n ouly-hrge Brykinin frgment 1-9, respetively, t 2 M onentrtion n vrying potentil ifferenes on triple qurupole n Fourier trnsform ion ylotron resonne (FT-ICR) mss spetrometer. On the triple qurupole instrument, the rithmeti men for ouly-hrge Brykinin frgment 2-9 slightly inrese with inresing potentil ifferene until mximum ws rehe t 2 V, followe y shrp erese t higher vlues, wheres for ouly-hrge Brykinin frgment 1-9 the sme tren with mximum t 17 V ourre (Figure 7). The stnr evition for ouly-hrge Brykinin frgment 2-9 initilly inrese with inresing potentil ifferenes, rehe mximum t 2V, then erese gin, whih ws lso oserve for oulyhrge Brykinin frgment 1-9 with the mximum shifte to 25 V (Figure 7). By ontrst, for the unproesse frgment spetr of ouly-hrge Brykinin frgment 2-9 tht were Ion Ativtion Attenution (B) Ion Ativtion Attenution (B) Figure 7. Depenene of (, ) rithmeti men n (, ) stnr evition on (, ) the potentil ifferene of triple qurupole instrument for ouly-hrge Brykinin 2-9 peptie (imons) n ouly-hrge Brykinin 1-9 peptie (squres), n on (, ) the ion tivtion ttenution of n FT-ICR instrument for oulyhrge Brykinin 2-9 peptie when proessing rw t (tringles) n t fter noise filtering (imons) s well s for oulyhrge Brykinin 1-9 peptie fter noise filtering (squres).

9 J Am So Mss Spetrom 25, 16, STATISTICAL EVALUATION OF PEPTIDE TANDEM MASS SPECTRA 513 quire on the FT-ICR instrument, oth rithmeti men (Figure 7) n stnr evition (Figure 7) remine virtully unhnge over the entire rnge. Closer inspetion of the spetr showe tht they onsist lrgely of rnom noise s kgroun to whih few frgment ion signls re superimpose. Consequently, we proesse eh spetrum to remove the noise using wvelet enoising funtion [29] followe y seline orretion. We then lulte rithmeti men (Figure 7) n stnr evition (Figure 7) for the filtere frgment spetr n plotte them ginst eresing ion tivtion ttenution on the x-xis, whih orrespons to inresing kineti energy of the preursor ions. The rithmeti men showe slight erese with eresing ttenution, whih eme muh steeper t vlue of 28 B efore flttening. By ontrst, the stnr evition showe mximum t vlue of 27 B while it ws signifintly lower t higher n lower ttenution. For ouly-hrge Brykinin frgment 1-9, whih ws hosen s seon exmple, similr trens n mxim with only minor hnges in shpe were oserve, whih my e initive of the ifferent experimentl setup when performing preursor isoltion, ollision-inue frgmenttion, n frgment spetr quisition in n ICR ell. In generl, the resulting plots for rithmeti men n stnr evition otine for oth instruments were inee omprle to those otine on hyri qurupole-time of flight instrument (Figures 2, 3), proving tht the metho ws not limite to speifi type of instrument. Applition for Clirtion Plots Finlly, we eie to emonstrte the ppliility of this metho for the existing rolling ollision energy pproh [13, 24]. In this setup, speifi potentil ifferene is use for eh mss-to-hrge rtio ssuming liner epenene. Slope n interept of this eqution re preefine n hve to e etermine using stnr pepties. Using trypti in-solution igests of BSA, -sein, n fetuin t 2 M onentrtion eh, we selete 22 ifferent ouly-hrge preursor peptie ions for frgmenttion n vrie the potentil ifferene in inrements of 2 V. For eh peptie, rithmeti men n stnr evition were lulte n plotte ginst the potentil ifferene. The mximl stnr evitions were then plotte ginst the mss-to-hrge rtios of the iniviul peptie preursor ions (Figure 8). The grph showe tht eh peptie h unique potentil ifferene tht resulte in mximl stnr evition, whih oul e pproximte y est-fit funtion using liner regression. Closer inspetion revele tht most vlues i not fll on the line ut iffere y s muh s 5 to 1 V from their neighors. Aitionl sttistil nlysis revele tht 15 out of 22 pepties h mximum vlues within one stnr evition of the est-fit line, wheres n itionl six pepties were lote etween one n two stnr evitions. Similr results were otine for 6 Potentil Differene t Mximum (V) m/z 7 9 Figure 8. Depenene of the optiml potentil ifferene etermine y the stnr evition pproh on the mss-to-hrge rtio of 22 ouly-hrge pepties present in trypti igests of BSA, -sein, n fetuin on hyri qurupole-time of flight instrument. Liner regression resulte in est-fit funtion (soli line) s well s res efine y one (she line) n two (otte line) stnr evition limits. triply-hrge peptie ions (t not shown), initing tht the metho is lso pplile to unknown pepties provie the hrge stte n e ientifie orretly. We onlue tht the slope n interept of the est-fit line etermine y the stnr evition metho efines the enter in the rolling ollision energy pproh, wheres the mximum istne of vlue from this line oul e use to etermine the spre of the ollision energy. Using istne of two stnr evitions of the est-fit line, whih orrespons to 6.2 V n ontins 21 out of 22 pepties in this experiment, will gurntee mximum frgment ion overge in t lest one of the settings tht re pplie for the vst mjority of pepties. Consequently, the sttistil metho presente here n e use immeitely to optimize iniviul settings for mximl frgment ion overge on vriety of instruments. Conlusions The results reporte here lerly show tht lultion of rithmeti men n stnr evition lens itself to the evlution of peptie frgment spetr for ifferent types of pepties, instruments, n onitions. Therefore, this sttistil metho n e use off-line to optimize iniviul settings for mximl frgment ion overge. On-line pplitions to optimize the ollision energy for eh peptie on n LC-MS/MS timesle woul require signifint hnges in instrument ontrol softwre n hve not een pursue, ut might well e fesile.

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