Hi3 Standards 1

[ CARE AND USE MANUAL ]

I . INT RODUCT IONThe Hi3 Ecoli and Phos B standards are a specialized set of 6

synthetically prepared and highly purified peptides that provide

the top 6 ionizing peptides of both the E.coli ClpB and rabbit

Phosphorylase B proteins. These standards are quantitated via

AAA to equimolar ratios to provide greater accuracy to each

analysis. They are greater than 95% pure and have been verified

by mass identification. In addition, each vial is in a Waters Max

Recovery vial 186000327C which simplifies the sample

preparation process.

These standards are primarily intended for use with the Hi3

quantification method for MSE proteomics data processed with

ProteinLynx Global Server™. The E. coli standard is intended for

samples of animal origin, and the Phos B standard is intended for

samples of microbial origin. The standards may also be of use in the

evaluation and benchmarking of proteomic LC/MS systems comprised

of nanoACQUITY UPLC® and Synapt® and Xevo® time-of-flight

mass spectrometers.

Table 1: Hi3 Phos B Standard (P/N 186006011)

Peptide Sequence

Average Molecular

WeightConcentration

Phos B Sequence 1

VLYPNDNFFEGK 1442.5913 1nmol/vial

Phos B Sequence 2

TC*AYTNHTVLPEALER 1875.0931 1nmol/vial

Phos B Sequence 3

IGEEYISDLDQLRK 1678.8602 1nmol/vial

Phos B Sequence 4

LLSYVDDEAFIR 1440.6164 1nmol/vial

Phos B Sequence 5

LITAIGDVVNHDPVVGDR 1890.1273 1nmol/vial

Phos B Sequence 6

VFADYEEYVK 1262.3817 1nmol/vial

The cysteine in sequence 2 is carbamidomethylated.

Hi3 STANDARDS

Table 2: Hi3 EColi Standard (P/N: 186006012)

Peptide Sequence

Average Molecular

WeightConcentration

Ecoli ClpB Sequence 1

VIGQNEAVDAVSNAIR 1655.8292 1nmol/vial

Ecoli ClpB Sequence 2

NNPVLIGEPGVGK 1293.4863 1nmol/vial

Ecoli ClpB Sequence 3

AIDLIDEAASSIR 1373.5266 1nmol/vial

Ecoli ClpB Sequence 4

VTDAEIAEVLAR 1286.4484 1nmol/vial

Ecoli ClpB Sequence 5

AIQQQIENPLAQQILSGELVPGK 2474.8412 1nmol/vial

Ecoli ClpB Sequence 6

LPQVEGTGGDVQPSQDLVR 1995.1776 1nmol/vial

I I . STORAGE AND STABILITY

Lyophilized peptides generally have excellent stabilities, often

showing little or no degradation after a few years at -20 °C. Long

term storage (>1 year) should be at -80 °C desiccated, medium

term storage (1-12 months) should be at -20 °C desiccated, short

term storage (<1 month) may be at 4 °C. Once reconstituted the

solution should be used immediately to avoid degradation of

peptides which would compromise the quantitative benefit.

I I I . RECONSTITUTION OF THE Hi3 DIGESTION STANDARDS

The Hi3 Digestion Standards contain 1 nmol of each of the peptides

listed in Table 1 or 2. It is recommended that 100 µL of 0.5%

trifluoroacetic acid in 3% acetonitrile/water be added to a vial of

standard to obtain a 10 pmol/µL stock solution. Because peptides can

adhere to glass and other surfaces resulting in significant losses from

highly dilute solutions, further dilutions should be made just prior to

addition to samples and excess diluted standard should be discarded.

Unused stock solutions may be stored in a freezer for a brief time.

Analysis of small amounts of standard (ca. 50 fmol) should only be

carried out in the presence of a sample matrix such as E. coli digest

of a serum digest (60 ng/µL or greater).

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/MSE 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:

Hi3 Standards 3

[ CARE AND USE MANUAL ]

Process and search the data in the normal fashion. Calculated

quantities for each identified protein (in fmol and ng) will be

found in the rightmost two columns of the protein results table

in the search result window (below):

Refer to IdentityE and ProteinLynx Global Server documentation

for further information.

MSDS information: T he Hi3 standards contain various non-

hazardous peptides in powder form. They are for laboratory use only;

solely for purposes of scientific experimentation, analysis, research

or development. These standards should be used only as directed

and in accordance with good laboratory practice. These standards

are non hazardous as defined by OSHA’s Hazard Communication

Standard, 29 CFR 1910.1200(c) and Appendix A and B, and are

thus exempt from the requirements of the Hazard Communication

Standard, including the requirement to supply MSDSs.

V. ORDERING INFORMATION

Part Number Product Description Qty

186002326MassPREP™ Digestion Standard,

Phosphorylase B1/pk

186003196MassPREP Ecoli Digestion

Standard1/pk

186006011 Hi3 Phos B Standard 1/pk

186006012 Hi3 Ecoli Standard 1/pk

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

Hi3 Standards 4

[ CARE AND USE MANUAL ]

©2012 Waters Corporation. Waters, UPLC, nanoACQUITY UPLC, Synapt, and Xevo are registered trademarks of Waters Corporation. The Science of What's Possible, MassPrep and ProteinLynx Global Server are trademarks of Waters Corporation. All other trademarks are the property of their respective owners.

March 2012 720004332EN IH-PDF

Austria and European Export (Central South Eastern Europe, CIS and Middle East) 43 1 877 18 07

Australia 61 2 9933 1777

Belgium 32 2 726 1000

Brazil 55 11 5094-3788

Canada 1 800 252 4752 x2205

China 86 21 6879 5888

CIS/Russia +497 727 4490/290 9737

Czech Republic 420 2 617 1 1384

Denmark 45 46 59 8080

Finland 09 5659 6288

France 33 1 30 48 72 00

Germany 49 6196 400600

Hong Kong 852 2964 1800

Hungary 36 1 350 5086

India and India Subcontinent 91 80 2837 1900

Ireland 353 1 448 1500

Italy 39 02 265 0983

Japan 81 3 3471 7191

Korea 82 2 6300 4800

Mexico 52 55 5524 7636

The Netherlands 31 76 508 7200

Norway 47 6 384 60 50

Poland 48 22 6393000

Puerto Rico 1 787 747 8445

Singapore 86 21 6879 5888

Spain 34 936 009 300

Sweden 46 8 555 11 500

Switzerland 41 56 676 70 00

Taiwan 886 2 2543 1898

United Kingdom 44 208 238 6100

All other countries: Waters Corporation U.S.A. 1 508 478 2000

1 800 252 4752

www.waters.com