Waters Hi3 Standards - Manual (Page 2)

Hi3 Standards 2
[ CARE AND USE MANUAL ]
IV. USING THE Hi3 DIGESTION STANDARDS
FOR QUANTIFICATION
For a typical proteomic analysis carried out with 75 µ i.d. UPLC ®
Columns, sufficient Hi3 standard should be added to each sample to
provide ca. 50 fmol of standard per injection. The injected quantity
should be scaled for other column diameters. LC/MS
E data should be
obtained in the normal manner.
For data processing, first append the appropriate protein sequence
below to the databank to be searched for protein identification
using a text editor.
>CLPB_ECOLI (P03815) ClpB protein (Heat shock protein F84.1)
MRLDRLTNKFQLALADAQSLALGHDNQFIEPLHLMSALLNQEGGSVS
PLLTSAGINAGQLRTDINQALNRLPQVEGTGGDVQPSQDLVRVLNLCDK
LAQKRGDNFISSELFVLAALESRGTLADILKAAGATTANITQAIEQMRG
GESVNDQGAEDQRQALKKYTIDLTERAEQGKLDPVIGRDEEIRRTIQVL
QRRTKNNPVLIGEPGVGKTAIVEGLAQRIINGEVPEGLKGRRVLALDMG
ALVAGAKYRGEFEERLKGVLNDLAKQEGNVILFIDELHTMVGAGKADG
AMDAGNMLKPALARGELHCVGATTLDEYRQYIEKDAALERRFQKVFV
AEPSVEDTIAILRGLKERYELHHHVQITDPAIVAAATLSHRYIADRQLP
DKAIDLIDEAASSIRMQIDSKPEELDRLDRRIIQLKLEQQALMKESDEAS
KKRLDMLNEELSDKERQYSELEEEWKAEKASLSGTQTIKAELEQAKIAI
EQARRVGDLARMSELQYGKIPELEKQLEAATQLEGKTMRLLRNKVTDA
EIAEVLARWTGIPVSRMMESEREKLLRMEQELHHRVIGQNEAVDAVSN
AIRRSRAGLADPNRPIGSFLFLGPTGVGKTELCKALANFMFDSDEAMV
RIDMSEFMEKHSVSRLVGAPPGYVGYEEGGYLTEAVRRRPYSVILLDEV
EKAHPDVFNILLQVLDDGRLTDGQGRTVDFRNTVVIMTSNLGSDLIQER
FGELDYAHMKELVLGVVSHNFRPEFINRIDEVVVFHPLGEQHIASIAQIQ
LKRLYKRLEERGYEIHISDEALKLLSENGYDPVYGARPLKRAIQQQIENP
LAQQILSGELVPGKVIRLEVNEDRIVAVQ
>PHS2_RABIT (P00489) GLYCOGEN PHOSPHORYLASE, MUSCLE
FORM (ECSRPLSDQEKRKQISVRGLAGVENVTELKKNFNRHLHFTLVK
DRNVATPRDYYFALAHTVRDHLVGRWIRTQQHYYEKDPKRIYYLSLE
FYMGRTLQNTMVNLALENACDEATYQLGLDMEELEEIEEDAGLGNGGL
GRLAACFLDSMATLGLAAYGYGIRYEFGIFNQKICGGWQMEEADDWL
RYGNPWEKARPEFTLPVHFYGRVEHTSQGAKWVDTQVVLAMPYDTP
VPGYRNNVVNTMRLWSAKAPNDFNLKDFNVGGYIQAVLDRNLAENIS
RVLYPNDNFFEGKELRLKQEYFVVAATLQDIIRRFKSSKFGCRDPVRTN
FDAFPDKVAIQLNDTHPSLAIPELMRVLVDLERLDWDKAWEVTVKTCA
YTNHTVLPEALERWPVHLLETLLPRHLQIIYEINQRFLNRVAAAFPGDV
DRLRRMSLVEEGAVKRINMAHLCIAGSHAVNGVARIHSEILKKTIFKDFY
ELEPHKFQNKTNGITPRRWLVLCNPGLAEIIAERIGEEYISDLDQLRKLLS
YVDDEAFIRDVAKVKQENKLKFAAYLEREYKVHINPNSLFDVQVKRIH
EYKRQLLNCLHVITLYNRIKKEPNKFVVPRTVMIGGKAAPGYHMAKMII
KLITAIGDVVNHDPVVGDRLRVIFLENYRVSLAEKVIPAADLSEQISTAG
TEASGTGNMKFMLNGALTIGTMDGANVEMAEEAGEENFFIFGMRVEDV
DRLDQRGYNAQEYYDRIPELRQIIEQLSSGFFSPKQPDLFKDIVNMLMH
HDRFKVFADYEEYVKCQERVSALYKNPREWTRMVIRNIATSGKFSSDR
TIAQYAREIWGVEPSRQRLPAPDEKIP
Configure the modified databank with the PLGS Databank Admin Tool.
Create a search workflow using the PLGS Workflow Designer,
being sure that the modified databank is selected, the proper
identifier for the standard protein is entered on the Calibration
Protein line and the number of femtomoles injected is entered
on the Calibration Protein Concentration line, as below: