Agilent G3314AA BioConfirm Software

Transcription

1 Agilent G3314AA BioConfirm Softwre Quik Strt Guide Use this guide to instll nd get strted with the BioConfirm softwre. Wht is BioConfirm Softwre? Agilent G3314AA BioConfirm Softwre lets you onfirm the identity of the proteins, syntheti peptides or peptides in your smples y mthing theoretil msses with the mesured msses in TOF mss spetrl dt. You ess ll of the following pilities through seprte pplitions in the Agilent TOF Softwre folder: Set up worklists for utomti identity onfirmtion of proteins, syntheti peptides nd protein digests. You enter method nd moleulr weight or sequene file (.psq) for eh smple. This dd-on pility is integrted into the Agilent TOF softwre. Set up methods to use in worklists to utomtilly onfirm the identity of proteins, syntheti peptides nd protein digests. This pility is integrted into the Dt Anlysis Method Editor. Crete nd modify sequenes to enter into the worklist s.psq files through the Sequene Editor - Mther. Run omprison of theoretil msses of sequenes for syntheti peptides or protein digest peptides with the mesured msses from TOF dt through the Sequene Editor - Mther. Intertively review the dt to onfirm the presene of proteins in smple through the Protein pplition. The Agilent G3314AA BioConfirm Softwre is supported with version A of the Agilent TOF softwre. Agilent Tehnologies

2 Differenes etween Protein Confirmtion softwre nd BioConfirm softwre The differenes etween the originl Protein Confirmtion softwre nd the BioConfirm softwre re desried in the tle elow: Tle 1 Differenes etween Protein Confirmtion softwre nd BioConfirm softwre Lotion in softwre of differenes Protein Confirmtion softwre BioConfirm softwre Dt Anlysis Method Editor methods for worklists TOF softwre min window worklists for utomted onfirmtion of identity Crete methods for proteins only (Protein Anlysis t) Enter protein methods only Must enter Smple Nme for eh smple Enter moleulr weight or sequene itself for eh smple into new Protein olumn From Add Column(s) dilog ox n ess Protein Editor to rete/edit sequenes Crete methods for proteins (Protein Anlysis t), peptides nd protein digests (Peptide/Protein Digest t) Cn enter methods for proteins, peptides or protein digests Cn leve Smple Nme lnk Enter moleulr weight (for proteins or syntheti peptides) or sequene file (.psq) into new Protein olumn From Add Column(s) dilog ox nnot ess Editor to rete/edit sequenes Cn run old protein worklists with originl ell sequenes Editor for reting/editing sequenes Protein Editor Proteins only Funtions essile through ts nd uttons Sequene sved s.xml file Sequene Editor - Mther Proteins, peptides nd protein digests Funtions essile through menus Sequene sved s.psq file for esy use with worklist Cn run omprison etween theoretil msses for syntheti peptides nd digest peptides with the msses from TOF dt Protein pplition (essile from Agilent TOF group folder) No differenes for proteins only 2 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

3 Instlling the TOF Protein Confirmtion softwre Step 1 Prepre to instll the softwre 1 Mke sure tht version A of the Agilent TOF softwre is instlled. CAUTION Do not strt the Agilent TOF softwre until the BioConfirm softwre is instlled. The Agilent TOF softwre engines must e stopped efore you instll the BioConfirm softwre. 2 Doule-lik the Agilent TOF softwre folder. 3 Doule-lik the System Lunher ion. 4 If the engines re running, lik Shutdown. 5 Wit for the engines to stop, nd lik Close. 6 When you re ertin the engines re stopped, instll the BioConfirm softwre. Step 2 Instll the BioConfirm softwre 1 Expnd the G3314AA BioConfirm folder on the CD, nd doule-lik setup.exe. 2 Clik Next to ring up the liense greement. 3 Clik Yes to ept the liense greement. The softwre instlls. 4 Clik Finish. You do not hve to reoot to strt the softwre. Step 3-Copy the exmple dt files from the instlltion CD 1 Open the Exmple Dt Files folder on the CD. 2 Copy ll of the.wiff files in the dt folder into the defult dt pth, usully \\PE Siex Dt\Projets\Defult\Dt. 3 Copy ll of the.psq files from the ProteinSequenes folder into \\Progrm Files\Agilent\TOF Softwre\ProteinSequenes folder. To uninstll the softwre Use the Add/Remove opertion of the Control Pnel to uninstll the softwre. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 3

4 Getting strted with the BioConfirm softwre After instlltion of the BioConfirm softwre, dditionl ions representing different protein pplitions pper in the Agilent TOF softwre folder. Figure 1 Agilent TOF Softwre group folder You use the new pplitions tht pper in the Agilent TOF folder, s well s new funtions inorported into the TOF softwre, to onfirm the identity of the proteins, syntheti peptides or protein digest peptides in your smples. On the next pge re tles tht list the exerises in this guide to help you perform the following tsks: Confirm protein identity utomtilly Confirm peptide identity utomtilly (syntheti peptides or protein digest peptides) Confirm protein/peptide/protein digest identity intertively 4 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

5 .. Tle 2 Confirm protein identity utomtilly If you wnt to do this: Set up method for utomted protein onfirmtion. Crete or edit protein sequene. Then doule-lik this ion in the Agilent TOF softwre folder: Dt Anlysis Method Editor Refer to this exerise: Exerise 1 Set up method for utomted protein onfirmtion on pge 7 Exerise 2 Crete protein sequene on pge 9 Set up nd run worklist to utomte protein onfirmtion. Sequene Editor - Mther TOF Exerise 3 Set up worklist for utomted protein onfirmtion on pge 12 Tle 3 Confirm peptide identity utomtilly (syntheti peptides or digest peptides) If you wnt to do this: Set up method for utomted peptide/protein digest onfirmtion. Crete or edit protein digest or syntheti peptide sequene. Set up nd run worklist to utomte peptide/protein digest onfirmtion. Then doule-lik this ion in the Agilent TOF softwre folder: Dt Anlysis Method Editor Sequene Editor - Mther TOF Refer to this exerise: Exerise 4 Set up method for utomted peptide/digest onfirmtion on pge 14 Exerise 5 Crete protein digest or syntheti peptide sequene on pge 17 Exerise 6 Set up worklist for utomted peptide/digest onfirmtion on pge 21 Agilent G3314AA BioConfirm Softwre Quik Strt Guide 5

6 Tle 4 Confirm protein/peptide/protein digest identity intertively If you wnt to do this: Confirm peptide/protein digest identity intertively Confirm protein identity intertively Then doule-lik this ion in the Agilent TOF softwre folder: Sequene Editor - Mther Refer to this exerise: Exerise 7 Confirm peptide identity intertively (syntheti or digest peptides) on pge 23 Exerise 8 Confirm protein identity intertively on pge 26 Protein 6 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

7 Exerises: Confirming protein identity utomtilly Exerise 1 Set up method for utomted protein onfirmtion This exerise guides you through the setup of protein onfirmtion method for ytohrome, whih you will use to utomtilly onfirm its presene in n unknown smple in Exerise 3 Set up worklist for utomted protein onfirmtion on pge 12. You enter the prmeters tht the worklist uses to utomtilly selet the hromtogrm nd spetrl peks, perform deonvolution nd generte protein reports. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the Dt Anlysis Method Editor. Doule-lik the Agilent TOF Softwre folder on your desktop. In the Agilent TOF Softwre folder, doule-lik the Dt Anlysis Method Editor ion. If the Protein Anlysis t is ville, skip to step 3. When you first open the Method Editor fter instlling the BioConfirm softwre, you see only the Properties nd Formul Confirmtion ts. You must ring up the Protein Anlysis t nd remove the Formul Confirmtion t. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 7

8 Steps Detiled Instrutions Comments 2 To rete new method, open the method, protein_ig_tol.nm, whih is instlled with the softwre. Selet File > Open. Selet protein_ig_tol.nm, nd lik Open. To rete ny new protein method, use the defult methods tht ome with the softwre. These methods hve the Protein Anlysis t dded nd the Formul Confirmtion t removed. To dd or remove ny of the Dt Anlysis Method Editor ts, lik the Selet DA Opertions ion, or selet Edit > Selet DA Opertions. 3 Chnge the following Chromtogrm Option. Mss Rnge = Clik the Protein Anlysis t. Clik the Chromtogrm Options t For the Strt M/Z, enter 700. d For the Stop M/Z, enter Restriting the mss rnge improves the mss tolerne. 4 Sve the file s iii_yt_ in the dmethods folder, nd lose the method editor. iii represents your initils. d e Clik File > Sve As. Open the dmethods folder. Enter the nme iii_yt_, nd lik OK. Enter omment in the method logook tht ppers, nd lik OK. Selet File > Exit. The dmethods folder n usully e found in the \\Progrm Files\Agilent\TOF Softwre pth. 8 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

9 Exerise 2 Crete protein sequene In BioConfirm you rete or edit protein sequene file to use in worklist to utomte protein onfirmtion. You rete or edit the file in the Sequene Editor - Mther. This exerise guides you through the retion of ytohrome sequene file tht you will use in Exerise 3 Set up worklist for utomted protein onfirmtion on pge 12. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the Sequene Editor - Mther. Doule-lik the Agilent TOF Softwre folder on your desktop. In the Agilent TOF Softwre folder, doule-lik the Sequene Editor - Mther ion. You n either open n existing sequene file to edit, or enter or opy nd pste sequene into the sequene text ox. The lue numers to the left nd right of the sequene re the pleholders for Index numers to help lote mino id positions. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 9

10 Steps Detiled Instrutions Comments 2 Copy the following ytohrome sequene, nd pste it into the sequene text ox. GDVEKGKKIFVQK- CAQCHTVEKGGKHKTGPNLH- GLFGRKTGQAPGFSYTDANKNK GITWGEETLMEYLENPKKY- IPGTKMIF- AGIKKKGEREDLIAYLKKATNE d Clik the Selet Text utton in the top toolr of Adoe Reder. Highlight the sequene, nd lik the Copy ion. Ple the ursor in the sequene text ox etween the N-term symol nd the C-term symol, nd lik the Pste ion. For the Smple Nme, enter the nme ytohrome. The sequene for ytohrome ppers in the Sequene Editor - Mther fter psting. 10 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

11 Steps Detiled Instrutions Comments 3 Modify the sequene so tht: the N-terminus is etylted, nd the ysteine t position 14 nd the ysteine t position 17 re linked with heme. Ple the ursor over the N-term symol, nd right-lik. Selet Modifitions... > Aetyltion. Clik Apply. d Clik OK. e Selet Edit > Links,,,. f In From Index: enter 14. g In To Index: enter 17, h For Link type, selet Heme. i Clik Add. j Clik OK. After etyltion the N-term symol is red, Itliized nd old. The linked ysteines re red, old nd underlined. 4 To omplete the sequene file retion: Chnge the Smple Nme to iiiyt, where iii represents your initils. Sve the file s iiiyt in the ProteinSequenes folder. Close the Sequene Editor - Mther. d e Enter iiiyt s the Smple Nme. Clik File > Sve. Open the ProteinSequenes folder, if neessry. Enter the nme iiiyt, nd lik Sve. Selet File > Exit. The ProteinSequenes ws reted when the softwre ws instlled. You n usully find this folder in the \\Progrm Files\Agilent\TOF Softwre pth. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 11

12 Exerise 3 Set up worklist for utomted protein onfirmtion This exerise guides you through the setup of worklist to utomtilly onfirm the presene of ytohrome in previously quired smple. You set up worklist for the smple, using the method from Exerise 1 Set up method for utomted protein onfirmtion on pge 7 nd the sequene file from Exerise 2 Crete protein sequene on pge 9. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the TOF softwre. Doule-lik the Agilent TOF Softwre ion on the Desktop. Doule-lik the TOF ion. 2 Set up worklist of one smple. Protein onfirmtion method \\Defult folder\ dmethods\iii_yt_.nm Dt file \\Defult folder\ Dt\Cytohrome.wiff Hide ll other olumns. d Selet iii_yt_ s the DA method. Selet Cytohrome.wiff s the Dt File. Right-lik the upper left-hnd orner of the worklist pne, nd selet Show/hide olumns. Cler the Aquisition Method, Smple Position, Smple Type, Inj. Vol. nd Comment hek oxes, nd lik OK. You need to enter smple nme only when you wnt to run ny ut the first smple in dt file. The Defult folder for dt nlysis methods is usully \\Progrm Files\Agilent\TOF Softwre\dmethods. The Defult folder for dt is usully PE Siex Dt\Projets\ Defult\Dt 3 Add protein olumn lled Cytohrome, nd enter its sequene file. d Right-lik the upper left-hnd orner of the worklist, nd selet Add Column(s). Selet Protein nd enter the Column nme s Cytohrome. Selet the iiiyt.psq file s the Vlue. Clik OK. If the worklist ontins more thn one smple, you usully leve the Vlue field lnk, nd enter different vlue in eh smple ell. The worklist should now look like the one shown elow. If the hek ox is missing, use the ottom sroll r to find it. 12 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

13 Steps Detiled Instrutions Comments 4 Set up to run the worklist for dt nlysis only. The pth for the DA method is the dmethods folder, usully under the TOF Softwre folder. The pth for the dt file is your defult dt folder. Right-lik the upper left-hnd orner of the worklist pne, nd selet Worklist Run Prmeters. Selet DA Only s Prt of method to run. Selet the pths to find the DA method nd the dt file, nd lik OK. The defult dt nlysis method pth is \\Progrm Files\Agilent\TOF Softwre\dmethods The defult dt file pth is \\PE Siex Dt\Projets\Defult\Dt. You n lso open the Worklist Run Prmeters dilog ox from the Worklist menu. 5 Sve the worklist s iiiyt.wkl, where iii represents your initils. Sve the worklist in the worklists folder under the TOF Softwre folder. Selet File > Sve > Worklist. Go to the worklists folder under the TOF Softwre folder. Enter iiiyt.wkl, nd lik Sve. The defult Worklist pth is \\Progrm Files\Agilent\TOF Softwre\worklists. 6 Run the worklist nd review the Protein Report. Hint: Go to your defult Dt folder d e f g Clik Strt for the worklist. After the run hs suessfully ompleted, go to your Dt folder. Expnd the dreports folder. Expnd the ytohrome folder. Expnd the ytohrome 1pmol_1 folder. Expnd the Protein_dte_time folder. Doule-lik Protein Report.htm. The method used for this protein nlysis sends the report to folder. The omprison or mthing of the theoretil msses of the protein sequene with the tul TOF msses is done using the mximum entropy deonvolution lgorithm. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 13

14 Exerises: Confirming peptide/protein digest identity utomtilly Exerise 4 Set up method for utomted peptide/digest onfirmtion This exerise guides you through the setup of onfirmtion method for syntheti peptide or set of peptides from protein digest. You will use the methods you rete in this exerise with Exerise 6 Set up worklist for utomted peptide/digest onfirmtion on pge 21. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the Dt Anlysis Method Editor. Doule-lik the Agilent TOF Softwre folder on your desktop. In the Agilent TOF Softwre folder, doule-lik the Dt Anlysis Method Editor ion. If the Peptide/Protein Digest t is ville, skip to step 3. When you first open the Method Editor fter instlling the BioConfirm softwre, you see only the Properties nd Formul Confirmtion ts. If the method from the previous exerise is loded, the Protein Anlysis t is visile. You must ring up the Peptide/ Protein Digest t nd remove the Formul Confirmtion t or the Protein Anlysis t. 14 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

15 Steps Detiled Instrutions Comments 2 To rete new method, open the method, peptide_method.nm, whih is instlled with the softwre. Selet File > Open. Selet peptide_method.nm, nd lik Open. To rete ny new peptide method, use the defult methods tht ome with the softwre. These methods hve the Peptide/Protein Digest t dded nd other ts removed. To dd or remove ny of the Dt Anlysis Method Editor ts, lik the Selet DA Opertions ion, or selet Edit > Selet DA Opertions. 3 Chnge the following settings: Strt retention time: 0 min End retention time: 0.5 min Mss Aury: 10 Clik the Peptide/Protein Digest t. Clik the Find Peptides Options t. You enter the settings in this dilog ox to use for the Mss Hunter lgorithm. Enter the vlues in the left olumn for the Chromtogrphi rnge nd the Mss Aury. Unlike protein onfirmtion, whih uses mximum entropy deonvolution to ompre msses, BioConfirm softwre uses the Mss Hunter lgorithm to ompre theoretil msses for the reted syntheti peptide or protein digest sequene with mesured msses from the TOF dt. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 15

16 Steps Detiled Instrutions Comments 4 Sve the file s iii_synth_pep.nm in the dmethods folder, nd lose the method editor. iii represents your initils 5 Repet steps 3 nd 4 for protein digest method. Use the defult method, peptide_method.nm. Enter 10 for Mss Aury. Chnge the m/z rnge to: Sve the file s iii_prot_dig.nm. d d e f g Clik File > Sve As. Open the dmethods folder. Enter the nme iii_synth_pep, nd lik Sve. Selet File > Exit. Selet File > Open. Selet peptide_method.nm, nd lik Open. Clik the Peptide/Protein Digest t. Clik the Find Peptides Options t. Enter Step 3 vlues for the R.T. rnge nd the Mss Aury. Enter M/z from s 200 nd M/z to s 950. Repet step 4, ut use the nme, iii_prot_dig. 16 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

17 Exerise 5 Crete protein digest or syntheti peptide sequene In BioConfirm you n rete or edit sequene file for syntheti peptide or for protein digest to use in worklist to utomte peptide onfirmtion. You rete or edit the file in the Sequene Editor - Mther. This exerise guides you through the retion of two sequene files tht you will use in Exerise 6 Set up worklist for utomted peptide/digest onfirmtion on pge 21. The first is sequene file for protein digest; the seond is for syntheti peptide. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the Sequene Editor - Mther. Doule-lik the Agilent TOF Softwre folder on your desktop. In the Agilent TOF Softwre folder, doule-lik the Sequene Editor - Mther ion. You n either open n existing sequene file to edit, or enter or ut nd pste sequene into the edit text ox. The lue numers to the left nd right of the sequene re the the Index numers to help lote mino id positions. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 17

18 Steps Detiled Instrutions Comments 2 Copy the serotrnsferrin sequene elow, nd pste it into the sequene text ox. Highlight the sequene, nd lik the Copy ion. Ple the ursor in the sequene text ox etween the N-term nd C-term symols, nd lik the Pste ion. The sequene for serotrnsferrin ppers in the Sequene Editor - Mther. MRPAVRALLACAVLGLCLADPERTVRWCTISTHEANKCASFRENVLRILESGPFVSCVKKTSHMDCIKAIS NNEADAVTLDGGLVYEAGLKPNNLKPVVAEFHGTKDNPQTHYYAVAVVKKDTDFKLNELRGKKSCHTGLGR SAGWNIPMAKLYKELPDPQESIQRAAANFFSASCVPCADQSSFPKLCQLCAGKGTDKCACSNHEPYFGYSG AFKCLMEGAGDVAFVKHSTVFDNLPNPEDRKNYELLCGDNTRKSVDDYQECYLAMVPSHAVVARTVGGKED VIWELLNHAQEHFGKDKPDNFQLFQSPHGKDLLFKDSADGFLKIPSKMDFELYLGYEYVTALQNLRESKPP DSSKDECMVKWCAIGHQERTKCDRWSGFSGGAIECETAENTEECIAKIMKGEADAMSLDGGYLYIAGKCGL VPVLAENYKTEGESCKNTPEKGYLAVAVVKTSDANINWNNLKDKKSCHTAVDRTAGWNIPMGLLYSKINNC KFDEFFSAGCAPGSPRNSSLCALCIGSEKGTGKECVPNSNERYYGYTGAFRCLVEKGDVAFVKDQTVIQNT DGNNNEAWAKNLKKENFEVLCKDGTRKPVTDAENCHLARGPNHAVVSRKDKATCVEKILNKQQDDFGKSVT DCTSNFCLFQSNSKDLLFRDDTKCLASIAKKTYDSYLGDDYVRAMTNLRQCSTSKLLEACTFHKP 3 Before the digestion, modify the sequene so tht ll the ysteines re lkylted with iodoetmide. d Selet Opertions > Glol Modifitions... From the Glol Modifitions list, selet Alkyltion (Iodoetmide). Clik Apply. Clik OK. Note tht fter modifition, the ysteines re now red nd Itliized. 18 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

19 Steps Detiled Instrutions Comments 4 Chnge the smple type from Protein to Protein Digest, nd omplete the Protein Settings Wizrd tht ppers. Use trypsin s the leving regent for the digest. Assume 2 missed levges. Aept the seleted peptide mth rules. d For Smple Type, selet Protein Digest. The Peptide Settings Wizrd ppers. Selet Trypsin s the leving regent, nd enter 2 for the numer of Missed Clevges. Clik Next. Clik OK. The theoretil msses of the sequene frgments nd the tul msses re mthed sed on the peptide mth rules. A list of theoretilly derived peptides nd their msses ppers in the lower hlf of the sreen fter you pply the digestion onditions. Clevge points re designted with lue rrows. 5 Sve the file s iiiserotrns in the ProteinSequenes folder under your dt folder, where iii respresents yoru initils. Clik File > Sve. Go to the ProteinSequenes folder under your dt folder. Enter iiiserotrns, nd lik Sve. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 19

20 Steps Detiled Instrutions Comments 6 Crete new sequene for syntheti peptide with the following letters: HLLQFNKMIKFETR Selet File > New. Type the letters in the left olumn into the sequene text ox etween the N-term nd C-term symols. For the Smple Type, selet Syntheti Peptide. The Peptide Mth Rules dilog ox ppers. 7 Ensure tht ll five peptide mth rules re seleted. Mke sure tht ll five peptide mth rules re seleted, nd lik OK. 8 Sve the file s iiisynthpep in the ProteinSequenes folder, where iii respresents your initils. Clik File > Sve. Go to the ProteinSequenes folder. Enter iiisynthpep, nd lik OK. 20 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

21 Exerise 6 Set up worklist for utomted peptide/digest onfirmtion This exerise guides you through the setup of worklist to utomtilly onfirm the presene of peptides in previously quired smple. You set up worklist for two smples, one for syntheti peptide nd one for digest of the protein serotrnsferrin. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the TOF softwre. Doule-lik the Agilent TOF Softwre ion on the Desktop. Doule-lik the TOF ion. 2 Set up worklist of 2 smples: Dt nlysis methods \\Defult folder\\dmethods \iii_synth_pep.nm nd iii_prot_dig.nm, respetively Dt files \\Defult folder\dt\synpep3.wiff nd Serotrnsferrin.wiff, respetively Hide ll other olumns d e Selet iii_synth_pep.nm s the DA method. Selet SynPep3.wiff s the Dt File. Repet steps nd for the protein digest informtion. Right-lik the upper left-hnd orner of the worklist pne, nd selet Show/hide olumns. Cler the Aquisition Method, Smple Position, Smple Type, Inj. Vol. nd Comment hek oxes, nd lik OK. You need to enter smple nme only when you wnt to run ny ut the first smple in dt file. If the hek ox is missing in the worklist, use the ottom sroll r to find it. 3 Add one protein olumns lled Peptides. Leve the Vlue fields lnk. For the first row, selet the iiisynthpep sequene file for the Peptides olumn. For the seond row, selet the iiiserotrns sequene file for the Peptides olumn. d e f Right-lik the upper left-hnd orner of the worklist, nd selet Add Column(s). Selet Protein nd enter the Column nme s Peptides. Clik OK. Right-lik on the worklist row nd selet Add Smple. Selet iii_prot_dig.nm s the DA method in the seond row. Selet the pproprite sequene files for eh row nd olumn. If the worklist ontins more thn one smple, you usully leve the Vlue field lnk, nd enter different vlue in eh smple ell. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 21

22 Steps Detiled Instrutions Comments The worklist should now look like the one shown elow. If the hek ox is missing, use the ottom sroll r to find it. 4 Set up to run the worklist for dt nlysis only. Use your defult pths for the dmethods nd the dt. Right-lik the upper left-hnd orner of the worklist pne, nd selet Worklist Run Prmeters. Selet DA Only s Prt of method to run. Selet the pths to find the DA method nd the dt file, nd lik OK. The defult method folder is usully \\Progrm Files\Agilent\ TOF Softwre\dmethods. The defult dt folder is usully \\PE Siex Dt\Projets\ Defult\Dt. 5 Sve the worklist s iiisynpepprotdig.wkl, where iii represents your initils. Selet File > Sve > Worklist. Enter iiisympepprotdig.wkl, nd lik Sve. Sve the worklist in the Worklists folder under your defult TOF softwre folder. 6 Run the worklist nd review the Protein Report. Hint: Go to your defult dt folder. d e Clik Strt for the worklist. After the run hs suessfully ompleted, go to the Dt folder under the TOF softwre folder. Expnd the dreports folder. Expnd the SynPep3 folder until you n see individul files, then doule-lik the.htm file. Expnd Serotrnsferrin folder until you n see individul files, then doule-lik the.htm file. The method used for this peptide nlysis sends the report to folder. 22 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

23 Exerises: Confirming protein/peptide/digest identity intertively Exerise 7 Confirm peptide identity intertively (syntheti or digest peptides) In BioConfirm you n set up the Sequene Editor - Mther to mth the theoretil msses of the syntheti peptide or protein digest peptides with the mesured msses in the TOF dt. This exerise shows you how to perform these mthes on the syntheti peptide you nd serotrnsferrin digest you reted in Exerise 5 Crete protein digest or syntheti peptide sequene on pge 17. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the Sequene Editor - Mther. Doule-lik the Agilent TOF Softwre folder on your desktop. In the Agilent TOF Softwre folder, doule-lik the Sequene Editor - Mther ion. In this exerise you open n lredy existing sequene file. 2 Open the protein digest sequene file. Selet File > Open. Selet iiiserotrns.psq, nd lik Open. The sequene for serotrnsferrin ppers in the Sequene Editor - Mther. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 23

24 Steps Detiled Instrutions Comments 3 Lod the TOF dt file Serotrnsferrin.wiff file. Selet File > Lod TOF dt. Selet the Serotrnsferrin.wiff file, nd lik Open. The Find Peptides dilog ox ppers. 4 Chnge the Find Peptides settings if neessry. Mss Aury = 10 ppm Chnge the Mss Aury to 10ppm. Clik OK. 5 View the mthed results, nd sve the file. Selet File > Sve. Note the mthes tht pper elow the sequene. 24 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

25 Steps Detiled Instrutions Comments 6 Repet steps 2-5 for the syntheti peptide. Open the sequene file iiisynthpep.psq Lod the TOF dt file SynPep3.wiff. Mss Aury = 5 Selet File > Sve. Note the mthes tht pper elow the sequene. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 25

26 Exerise 8 Confirm protein identity intertively This exerise guides you through the intertive viewing of dt from etltogloulin dt file. You ring up nd view the hromtogrm nd the spetrl peks over time rnge. You then deonvolute the spetrl peks to produe mss grph. From the mss grph, you generte list of proteins possily present in the smple nd deonvoluted ion set. Try the steps on the left without the detiled instrutions. If you need more help, follow the detiled instrutions. Steps Detiled Instrutions Comments 1 Strt the Protein Confirmtion softwre. Doule-lik the Agilent TOF Softwre folder on the Desktop. Doule-lik the Protein ion. You now see the Protein Confirmtion min window. 2 Open the dt file BetL.wiff under the Dt folder in your defult folder. Selet File > Open. Go up the folder tree to find the Dt folder. Selet BetL.wiff, nd lik Open. The Chromtogrm pne ppers. 26 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

27 Steps Detiled Instrutions Comments 3 Selet spetr from the hromtogrm over the 3.5 to 3.9 minute rnge. Drg the ursor over the rnge of minutes. Right-lik the hromtogrm, nd selet Show Spetrum. 4 Chnge deonvolution settings to those elow, nd egin deonvolution: Set the mss rnge to Use limited m/z rnge of d Right-lik the Spetrum pne. Selet Deonvolution Settings. Enter the Strt mss s nd the Stop mss s Mrk the Use limited m/z rnge, nd enter e Clik Strt. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 27

28 Steps Detiled Instrutions Comments At the end of deonvolution, you see the results, or mss grph. 28 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

29 Steps Detiled Instrutions Comments 5 Show the protein list for the two protein peks in the mss grph t mu nd mu. Clik on mu nd drg the ursor to mu. Right-lik the mss grph nd selet Show Protein List. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 29

30 Steps Detiled Instrutions Comments 6 Show the deonvoluted ion sets for the proteins found etween nd mu in the mss grph. Mke sure to view ll three sets. An ion set is grph of the m/z vlues (+ hrges) lulted from the mesured mss of est hit or seleted protein fter deonvolution. Right-lik the shded portion of the mss grph, nd selet Show Deonvoluted Ion Sets. Right-lik on the title of the pne, nd selet the next ion set. Repet step for the third ion set. The deonvoluted ion sets pne ppers ove the mss grph with one ion set in view. The three ion sets for the three est hit proteins re ville for viewing. You n lso selet proteins from the Protein List nd selet Show Deonvoluted Ion Sets from the shortut menu. 30 Agilent G3314AA BioConfirm Softwre Quik Strt Guide

31 Steps Detiled Instrutions Comments 7 Show the deonvoluted ion sets list. Right-lik the deonvoluted ion sets pne, nd selet Show Deonvoluted Ion Sets List. Clik OK to lose. 8 Show the rw spetrum for the first ion set. Right-lik the deonvoluted ion sets pne, nd selet Show Rw Spetrum. Agilent G3314AA BioConfirm Softwre Quik Strt Guide 31

32 In this Book This ook introdues you to the BioConfirm Softwre for the onfirmtion of protein nd peptide identity through mss spetrl omprisons. Agilent Tehnologies, In Printed in USA First edition, Septemer 2005 *G * G Agilent Tehnologies