Standard & Tryptic Digest Guide

Table of Contents Standard

P/N

Page

General Standard Information

CTM Series

3

Small Molecule Std—4 Parabens

CTM/00001/01

4

Medium Molecule Std—5 Angiotensins

CTM/00001/02

5

Phosphopeptide Std—2 Peptides & their Phospho Analogs

CTM/00001/05

6

Large Molecule Std—5 Intact Proteins

CTM/00001/07

7

Tryptic Peptide Std—4 Tryptic Peptides

CTM/00001/08

8

Protein Tryptic Digest

P/N

General Protein Tryptic Digest Information

PTD Series

9

Bovine Beta Lactoglobulin

PTD/00001/14

10

Bovine Alpha Casein

PTD/00001/16

11

Bovine Carbonic Anhydrase

PTD/00001/11

12

Bovine Glutamate Dehydrogenase

PTD/00001/25

13

Bovine Serum Albumin

PTD/00001/15

14

Bovine Lactoperoxidase

PTD/00001/27

15

6 Bovine Tryptic Digest Equal Molar Mix

PTD/00001/63

16-17

6 Bovine Tryptic Digest Exponential Molar Mix

PTD/00001/64

18-21

Equine Cytochrome C

PTD/00001/12

22

Equine Apo-Myoglobin

PTD/00001/04

23

Human Alpha lactalbumin

PTD/00001/40

24

Human Glycoprotein

PTD/00001/41

25

Yeast Alcohol Dehydrogenase

PTD/00001/36

26

Yeast Enolase

PTD/00001/46

27

Page

Standard Test Mixtures General Information Michrom offers five different Standard Test Mixtures that can be utilized for calibration, trouble-shooting and/or method development. All Standards Test Mixtures consist of four to five analytes which provide the user with a nice sampling range of the specific type of analytes across an HPLC gradient. ♦ ♦ ♦ ♦ ♦

Each standard consists of 5 micrograms of each analyte concentrated to dryness. All standards are salt-free. All standards are detergent-free. Each standard comes with a certificate of analysis Dilute standard to the desired concentration with buffer of choice (Example: Add 0.5 mL of 94.9/5/0.1% H2O/Acetonitrile/TFA to obtain 10ng/µL)

More detailed information can be obtained on the example test certificates that are available on the following pages: Standard

P/N

Page

Small Molecule Std—4 Parabens

CTM/00001/01

4

Medium Molecule Std—5 Angiotensins

CTM/00001/02

5

Phosphopeptide Std—2 Peptides & their Phospho Analogs

CTM/00001/05

6

Large Molecule Std—5 Intact Proteins

CTM/00001/07

7

Tryptic Peptide Std—4 Tryptic Peptides

CTM/00001/08

8

Certificate of Analysis Lot # 080212 Small Molecule Test Mixture (CTM/00001/01) (4 Parabens) 1) 2) 3) 4)

Methyl Paraben (152 mw) Ethyl Paraben (166 mw) Propyl Paraben (180 mw) Butyl Paraben (194 mw)

Each vial contains 5.0 micrograms of each Paraben (concentrated to dryness) Dilute to 500 μL for 10 ng/μL (using 2% Acetonitrile & 0.005-0.1% ion-pairing acid of choice) Store at 4 C° (once test mix is in solution) A Solv: B Solv: Instrument: Column: Flow Rate: Gradient: Detection: Temperature: Amount:

2/98/0.1% Acetonitrile/H2O/TFA 90/10/0.1% Acetonitrile/H2O/TFA Paradigm MS4 HPLC Micro Magic Bullet Halo C18 250 μL/min 5-95%B in 4 min UV at 205nm Ambient 10 μL 10 ng/μL Small Molecule Test Mix

Certificate of Analysis Lot # 080506 Medium Molecule Test Mixture (CTM/00001/02)(5 Angiotensins) Each vial contains 5.0 micrograms of each Angiotensin (concentrated to dryness) Dilute to 500 μL for 10 ng/μL (using 2% Acetonitrile & 0.005-0.1% ion-pairing acid of choice) Store at 4 C° (once test mix is in solution) 050608_cp361270_-NewANG_5_1600V_120C_02

5/7/2008 11:04:19 AM

RT: 0.00 - 14.99

NL: 1.35E8 Base Peak MS 050608_cp36 1270_NewANG_5_ 1600V_120C _02

5.94

100

7.25

95

6.67

90 85 8.27

80 75 7.83

70

Relative Abundance

65 60 55 50 45 40 35 30 25 20 15

13.25

10 5

0.82 1.30 1.67

0 0

1

3.01 3.38

2

3

4.25 4.82 4

5

10.26 10.60 9.34 9.80

5.76 6

7 8 Time (min)

9

10

11

11.83

13.07 12.10

12

13

13.34

13.75 13.82 14

Peak # (mole. wt.) Sequence (Amino Acids) Peak 1 (mw 883.1)

ARG-VAL-TYR-VAL-HIS-PRO-ILE

Peak 2 (mw 917.1)

ARG-VAL-TYR-VAL-HIS-PRO-PHE

Peak 3 (mw 931)

ARG-VAL-TYR-ILE-HIS-PRO-PHE

Peak 4 (mw 1282)

ASP-ARG-VAL-TYR-VAL-HIS-PRO-PHE-HIS LEU

Peak 5 (mw 1296)

ASP-ARG-VAL-TYR-ILE-HIS-PRO-PHE-HIS-LEU

Quality Assurance Test Method HPLC

Paradigm MS4 MDLC

Column

CP3-61270-00 0.1 x 50mm Magic C18AQ 3μ 200Å

Flow Rate

1.0 μL/min

Solvent A

5/95/0.1% Acetonitrile/H2O/Formic Acid

Solvent B

95/5/0.1% Acetonitrile/H2O/Formic Acid

Gradient

5-45%B 0-10 minutes

Detection

LCQ Deca XP+ MS

Standard

100 fmol Medium Molecule Test Mix

Certificate of Analysis Lot # 050415TD05 Phosphopeptide Test Mixture (CTM/00001/05) (2 Peptides & their Phospho Analogs) Each vial contains 5.0 micrograms of each Analyte (concentrated to dryness) Dilute to 500 μL for 10 ng/μL (using 2% Acetonitrile & 0.005-0.1% ion-pairing acid of choice) Store at 4 C° (once test mix is in solution) Peptide

m.w.

Charge

Sequence (Amino Acids)

1

773

+2

Leu- Arg- Arg- Ala- Ser- Leu- Gly

1P

853

+1

Leu- Arg- Arg- Ala- pSer- Leu- Gly

2

1463

-2

Thr- Ser- Thr- Glu- Pro- Gln- Tyr- Gln- Pro- Gly- Glu- Asn- Leu

2P

1543

-3

Thr- Ser- Thr- Glu- Pro- Gln- pTyr- Gln- Pro- Gly- Glu- Asn- Leu

PEPTIDE 1 & 1P ON 2D TiO2-RP LC/MS 1D NanoRPLC MS Run 500 fmol Peptide 1 500fmol Peptide 1P 2D TiO2 pH3 Wash 500 fmol Peptide 1 500fmol Peptide 1P 2D TiO2 pH11 Elute 500 fmol Peptide 1 500fmol Peptide 1P

PEPTIDE 2 & 2P ON 2D TiO2-RP LC/MS 1D NanoRPLC MS Run 500 fmol Peptide 2 500fmol Peptide 2P 2D TiO2 pH3 Wash 500 fmol Peptide 2 500fmol Peptide 2P 2D TiO2 pH11 Elute 500 fmol Peptide 2 500fmol Peptide 2P

Certificate of Analysis Lot # 080625 Large Molecule Test Mixture (CTM/00001/07) (5 Proteins) Each vial contains 5.0 micrograms of each Protein (concentrated to dryness) Dilute to 500 μL for 10 ng/μL (using 2% Acetonitrile & 0.005-0.1% ion-pairing acid of choice) Store at 4 C° (once test mix is in solution) PEAK #

PROTEIN

MOLECULAR SPECIES WEIGHT (KD)

SIGMA PART #

SIGMA LOT #

1

Ribonuclease A

13.7 KD

Bovine

R-5125

074K7680

2

Insulin

5.7 KD

Bovine

I-5500

104K0697

3

Lysozyme

14.3 KD

Chicken

L-6876

111H7010

4

α-Lactalbumin

14.2 KD

Bovine

L-6010

035K7005

5

Apomyoglobin

16.9 KD

Equine

A-8673

087K6018

A Solv: B Solv: Instrument: Column: Flow Rate: Gradient: Detection:

2/98/0.1% Acetonitrile/H2O/TFA 90/10/0.1% Acetonitrile/H2O/TFA Paradigm MS4 MDLC 0.2 x 150 mm PLRP-S 5μ 300A 5 μL/min 5-95%B in 20 min UV at 205nm

Certificate of Analysis Lot # 080605 TP4 Tryptic Peptide Test Mixture (CTM/00001/08) Each vial contains 5.0 micrograms of each Peptide (concentrated to dryness) Dilute to 500 μL for 10 ng/μL (using 2% Acetonitrile & 0.005-0.1% ion-pairing acid of choice) Store at 4 C° (once test mix is in solution) Peak # (mole. wt.) Sequence (Amino Acids) Peak 1 (mw 869)

H-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-OH

Peak 2 (mw 877)

H-Tyr-Ile-Tyr-Gly-Ser-Phe-Lys-OH

Peak 3 (mw 1262)

H-Ile-Ser-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH

Peak 4 (mw 1353)

H-Gly-Phe-Val-Phe-Thr-Leu-Thr-Val-Pro-Ser-Glu-Arg-OH

060408_cp361271_newtp4std

6/5/2008 9:20:52 AM

RT: 0.00 - 29.98 SM: 7G

NL: 1.27E8 Base Peak MS 060408_cp 361271_ne wtp4std

20.36

Pressure = 1800 PSI

100 95 90 85 80 75

14.11

70

Relative Abundance

65

15.17

60 55 50 45 12.29

40 35 30 25 20 15 10 5

1.39 1.94

3.51 4.75

6.19

7.55 8.21 9.80 10.77

0

2

4

6

8

10

12

21.72

16.46 18.84 18.95

13.74

0

14 16 Time (min)

18

20

22

24.25 24.42 24

26

27.83 28.57 28

Quality Assurance Test Method HPLC

Paradigm MS4 MDLC

Column

CP3-61271-00 0.1 x 150mm Magic C18AQ 3μ 200Å

Flow Rate

0.5 μL/min

Solvent A

5/95/0.1% Acetonitrile/H2O/Formic Acid

Solvent B

95/5/0.1% Acetonitrile/H2O/Formic Acid

Gradient

5-45%B 0-20 minutes

Detection

LCQ Deca XP+ MS with ADVANCE

Standard

100 fmol TP4 Tryptic Peptide Test Mix

Protein Tryptic Digest General Information Michrom offers fourteen Protein Tryptic Digests including two complex mixtures that can be utilized for calibration, method development and validation in various LC/MS applications. ♦ ♦ ♦ ♦ ♦ ♦ ♦

Each Protein Tryptic Digest (except complex mixtures) consists of 500 picomoles Pure proteins are reduced with DTT, alkylated with IAA and digested with Trypsin Monitored by HPLC for complete digestion Quantitated by HPLC/UV Analysis Certified by LC/MS/MS and protein database searched for peptide coverage All standards are detergent-free Dilute standard to the desired concentration with buffer of choice (Example: Add 0.5 mL of 94.9/5/0.1% H2O/Acetonitrile/TFA to obtain 10ng/µL)

More detailed information can be obtained on the example test certificates that are available on the following pages: Protein Tryptic Digest

P/N

Page

Bovine Beta Lactoglobulin

PTD/00001/14

10

Bovine Alpha Casein

PTD/00001/16

11

Bovine Carbonic Anhydrase

PTD/00001/11

12

Bovine Glutamate Dehydrogenase

PTD/00001/25

13

Bovine Serum Albumin

PTD/00001/15

14

Bovine Lactoperoxidase

PTD/00001/27

15

6 Bovine Tryptic Digest Equal Molar Mix

PTD/00001/63

16-17

6 Bovine Tryptic Digest Exponential Molar Mix

PTD/00001/64

18-21

Equine Cytochrome C

PTD/00001/12

22

Equine Apo-Myoglobin

PTD/00001/04

23

Human Alpha lactalbumin

PTD/00001/40

24

Human Glycoprotein

PTD/00001/41

25

Yeast Alcohol Dehydrogenase

PTD/00001/36

26

Yeast Enolase

PTD/00001/46

27

Paradigm LC-MS/MS Certified Digest Test Certificate Bovine Beta Lactoglobulin (P/N PTD/00001/14) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MDLC - ADVANCE - LCQ MS 0.2 x 150mm 3u 200A Magic C18 99.9/0.1 H2O/FA 100% ACN 2.0 ul/min (2000 PSI) 5-35%B in 30min plus hold 80%B for 2min MS and MS/MS 200 fmol

Source:

Sigma P/N L3908 Sigma Lot # 097K7012 Chemistry: Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) Protein MW: 18.3 KD Digest Lot #: 080318TD-14 Accession #: gi|2194089|

LIVTQTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP EGDLEILLQK WENGECAQKK IIAEKTKIPA VFKIDALNEN KVLVLDTDYK KYLLFCMENS AEPEQSLVCQ CLVRTPEVDD EALEKFDKAL KALPMHIRLS FNPTQLEEQC HI

Beta_Lactal_200fmol_2

3/18/2008 3:50:13 PM

RT: 0.00 - 39.99 NL: 7.21E9 TIC MS Beta_Lacta l_200fmol_ 2

35.39

100

22.27

90

Relative Abundance

35.46

22.24

80

23.91

70

22.32

60

35.28 24.24 35.51 34.68

50 40 30

22.22

16.18

20 2.07 2.46 2.50

10

5.03 5.58

8.82

8.86

11.99 12.47 14.02 16.14 16.12 13.87 9.79

24.26

23.82

24.29

34.47

24.34 24.41

34.39

21.82

16.23 16.45

34.54

22.35

20.89 18.69 19.37

24.85

25.67

27.15

30.16

35.57 35.64 35.74 35.88 35.91

34.20 32.47 33.85

0 0

2

4

6

8

10

12

14

16

18

20 Time (min)

22

24

26

28

30

32

34

38.63 38.14 38.82

36

38

RT: 0.00 - 39.99 SM: 7G NL: 1.08E9

22.27

100

Base Peak MS Beta_Lacta l_200fmol_ 2

90

Relative Abundance

80 70 60 50 40 24.24 30 20

16.19

10 0.93 2.07

0 0

2

4.95 5.09 7.26 7.39 4

6

11.99

8.86 8

10

16.85

12.47 14.00 12

14

14.71 16

25.09

21.86 19.39 20.86 18

20 Time (min)

22

24

25.64 26

28.11 28

30.24 30

33.69 32

35.17 34

35.41 36

35.65

38.19 38

Paradigm LC-MS/MS Certified Digest Test Certificate Bovine Alpha Casein (P/N PTD/00001/16) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MDLC - ADVANCE - LCQ MS 0.2 x 150mm 3u 200A Magic C18 99.9/0.1 H2O/FA 100% ACN 2.0 ul/min (2000 PSI) 5-35%B in 30 plus hold 80%B for 2min MS and MS/MS 200 fmol

Source:

Sigma P/N C6780 Sigma Lot # 075K7425 Chemistry: Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) Protein MW: 24.5 KD Digest Lot #: 080318TD-16 Accession #: IPI00706094 (S1) & IPI00698843 (S2)

MK LLILTCLVAVALAR PK HPIK HQGLPQEVLNENLLR FFVAPFPEVFGK EK VNELSK DIGSpESpT EDQAMEDIK QMEAESpISpSpSpEEIVPNSpVEQK HIQK EDVPSER YLGYLEQLLR LK K YK VPQLEIVPNSpAEER LHSMK EGIHAQQK EPMIGVNQELAYFYPELFR QFYQLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEK TTMPLW

Alpha_Casein_200fmol_2

3/19/2008 10:50:21 AM

RT: 0.00 - 40.01

90 34.95

80 Relative Abundance

NL: 1.76E9

35.24

100

70

34.82

60

TIC MS Alpha_Cas ein_200fm ol_2

35.42 35.48

35.55

34.72 35.67

2.18

50

28.03

34.65

28.06

40 1.99

30

2.21

1.95

19.37

20

0.64

2.60

10

5.23 5.38 5.69 6.93 7.81

11.30 11.55

13.09 14.88 16.90

18.24 19.32

24.89

23.85

20.47 20.37

27.18

23.73

20.55

28.08

34.41

28.17 29.31 29.95

25.02 25.26

34.37

35.72 35.79 35.91 36.04

33.25 34.23

38.10 39.42

0 0

2

4

6

8

10

12

14

16

18

20 Time (min)

22

24

26

28

30

32

34

36

38

40

RT: 0.00 - 40.01 SM: 7G NL: 1.42E8

28.06

100

Base Peak MS Alpha_Cas ein_200fm ol_2

90

Relative Abundance

80 70 60 27.18

50 40 20.46

30 20

7.50

19.37

7.20

10 1.53 2.18

0 0

2

4.01 4

5.71 6

7.82 9.24 8

11.84 13.99 10

12

14.15 14.84 17.55 14

16

24.69 23.67 24.89

18.24

18

21.94

30.01

23.10 20 Time (min)

22

24

26

28

30

30.43

33.28 34.63 35.28 35.51 35.86 38.65 39.75

32

34

36

38

40

Paradigm LC-MS/MS Certified Digest Test Certificate Bovine Carbonic Anhydrase (P/N PTD/00001/11) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MDLC - ADVANCE - LCQ MS 0.2 x 150mm 3u 200A Magic C18 99.9/0.1 H2O/FA 100% ACN 2.0 ul/min (2000 PSI) 5-35%B in 30min plus hold 80%B for 2min MS and MS/MS 200 fmol

Source:

Sigma P/N C3934 Sigma Lot # 043K1394 Chemistry: Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) Protein MW: 29.2 KD Digest Lot #: 080318TD-11 Accession #: IPI00716246

SHHWGYGKHN GPEHWHKDFP IANGERQSPV DIDTKAVVQD PALKPLALVY GEATSRRMVN NGHSFNVEYD DSQDKAVLKD GPLTGTYRLV QFHFHWGSSD DQGSEHTVDR KKYAAELHLV HWNTKYGDFG TAAQQPDGLA VVGVFLKVGD ANPALQKVLD ALDSIKTKGK STDFPNFDPG SLLPNVLDYW TYPGSLTTPP LLESVTWIVL KEPISVSSQQ MLKFRTLNFN AEGEPELLML ANWRPAQPLK NRQVRGFPK Carbonic_Anhy_200fmol_1

3/19/2008 11:33:38 AM

RT: 0.00 - 39.97

34.61

90 80 Relative Abundance

NL: 1.07E10 TIC MS Carbonic_ Anhy_200f mol_1

34.70

100

34.27

70 60 50 34.15

40

34.78

27.17

30

35.25

20 2.09 2.70 1.99 1.79 4.18 4.75

10 0 0

2

4

7.95 8.37 8.46 6

8

11.15 13.40

10

12

14.35 16.18 16.44 17.70 14

16

18

21.68

19.47 20 Time (min)

23.92 22.02 24.55

22

24

34.09

27.24 27.10

27.31 29.39 30.49 32.70

26

28

30

35.34 35.69 36.07

34.02

32

34

36

38.51 38

RT: 0.00 - 39.97 SM: 7G 27.17

100

NL: 1.90E8

34.70

Base Peak MS Carbonic_ Anhy_200f mol_1

90

Relative Abundance

34.15

21.68

80 70 60

34.78

21.74

50 11.09

40

14.35

30 15.09

20 10 0.08

0 0

2.49 2

7.87

4.18 5.15 4

6

8.03

9.88 8.99

8

13.28

23.92

20.70

16.44 18.81 19.47

27.31

14.07

22.98

24.04

26.55

27.38

30.42

12.19 10

12

14

16

18

20 Time (min)

22

24

26

28

30

31.83 33.59 32

35.07 34

36

35.97

38.61 38

Paradigm LC-MS/MS Certified Digest Test Certificate Bovine Glutamate Dehydrogenase (P/N PTD/00001/25) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MDLC - ADVANCE - LCQ MS 0.2 x 150mm 3u 200A Magic C18 99.9/0.1 H2O/FA 100% ACN 2.0 ul/min (2000 PSI) 5-35%B in 30min plus hold 80%B for 2min MS and MS/MS 200 fmol

Source:

Sigma P/N G7882 Sigma Lot # 105K7353 Chemistry: Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) Protein MW: 55.5 KD Digest Lot #: 080318TD-25 Accession #: gi|118533|

ADREDDPNFF KMVEGFFDRG ASIVEDKLVE DLKTRQTQEQ KRNRVRGILR IIKPCNHVLS LSFPIRRDDG SWEVIEGYRA QHSHQRTPCK GGIRYSTDVS VDEVKALASL MTYKCAVVDV PFGGAKAGVK INPKNYTDED LEKITRRFTM ELAKKGFIGP GVDVPAPNMS TGEREMSWIA DTYASTIGHY DINAHACVTK PGISQGGIHG RISATGRGVF GHIENFIENA SYMSILGMTP GFGDKTFAVQ GFGNVGLHSM RYLHRFGAKC VAVGESDGSI WNPDGIDPKE LEDFKLQHGT ILGFPKAKIY EGSILEVDCD ILIPAASEKQ LTKSNAPRVK AKIIAEGANG PTTPQADKIF LERNIMVIPD LYLNAGGVTV SYFQILKNLN HVSYGRLTFK YERDSNYHLL MSVQESLERK FGKHGGTIPI VPTAEFQDRI SGASEKDIVH SGLAYTMERS ARQIMRTAMK YNLGLDLRTA AYVNAIEKVF RVYNEAGVTF T Glutamic_Dehydrogenase_200fmol_1

3/19/2008 2:17:02 PM

RT: 0.00 - 40.02 NL: 8.47E9 TIC MS Glutamic_ Dehydroge nase_200f mol_1

34.30

100 90

34.38

Relative Abundance

80 70 60 50 40 30

34.22

20

34.12

10

2.58

2.48

2.68

0 0

2

4

5.96 7.12 6

8.81

8.89 9.05

8

10

22.65 23.73 11.52 12

13.78

15.41

14

16.42

18.20

16

18

20.67 20 Time (min)

22

24

29.58 29.51 29.66 30.09

25.08 25.47 26.31 26

28

30

34.47 34.65

33.20 34.02 32

35.41 35.70

34

36

36.63

39.77

38

40

RT: 0.00 - 40.02 SM: 7G NL: 1.83E8

34.38

100

Base Peak MS Glutamic_ Dehydroge nase_200f mol_1

90

Relative Abundance

80 34.12

70 23.73

60 50

29.58 22.65

40

25.47

30

16.49

25.56

13.72

20 8.81

10 1.69

0 0

2

3.50 3.57 6.14 6.86 4

6

8

8.89

16.42 9.05 10.69

11.54 12.40

10

12

14.52 14

16

34.84

22.54 18.20 20.67 22.02 18.01 19.94 18

20 Time (min)

22

27.28 27.72 25.92 24

26

28

35.11 36.17 37.88

29.82 31.52 33.20 30

32

34

36

38

40

Paradigm LC-MS/MS Certified Digest Test Certificate Bovine Serum Albumin (P/N PTD/00001/15) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MDLC - LCQ Deca XP+ MS 0.2 x 150mm 3u 200A Magic C18AQ 99.9/0.1 H2O/FA 98.9/1.0/0.1 ACN/H2O/FA 2.0 ul/min (2000 PSI) 5-35%B in 30min plus hold 80%B for 2min MS and MS/MS 200 fmol

Source:

Sigma P/N A4378 Sigma Lot # 025K7540 Chemistry: Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) Protein MW: 69.3 KD Digest Lot #: 090403TD-15 Accession #: gi|1351907|

MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA Q:\Test Mixture QC\...\040309_bsaqc-2

4/3/2009 2:25:16 PM

RT: 0.00 - 39.98 SM: 7G

NL: 2.17E8 Base Peak MS 040309_bs aqc-2

32.98

100 95 90 85 80 75 70

Relative Abundance

65 60

32.94

55 17.45

50

33.02 33.05

27.11

45 40 21.19

16.09

35

27.08

11.30

30 16.07

25

11.27

12.22

20 2.56

15

2.53 1.70

10 5

15.99

11.06

6.72 4.80 6.67 4.17 6.50

1.65

16.02

6.75

8.11

12.24

8.63

17.95 18.02 21.15

25.57

27.03 32.91

27.14

22.73 25.55 22.80

18.24

12.79

33.24

21.20

11.39

32.69

28.38 28.39

25.48

32.66 29.90 32.31 32.21

13.82

0 0

2

4

6

8

10

12

14

16

18

20 22 Time (min)

24

26

28

30

32

33.27 33.30 34.26 34

36.83 36

39.36 38

Paradigm LC-MS/MS Certified Digest Test Certificate Bovine Lactoperoxidase (P/N PTD/00001/27) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MDLC - ADVANCE - LCQ MS 0.2 x 150mm 3u 200A Magic C18 99.9/0.1 H2O/FA 100% ACN 2.0 ul/min (2000 PSI) 5-35%B in 30min plus hold 80%B for 2min MS and MS/MS 200 fmol

Source:

Sigma P/N L2005 Sigma Lot # 085K3781 Chemistry: Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) Protein MW: 79.8 KD Digest Lot #: 090310TD-27 Accession #: gi|129823|

MWVCLQLPVF LASVTLFEVA ASDTIAQAAS TTTISDAVSK VKIQVNKAFL DSRTRLKTTL SSEAPTTQQL SEYFKHAKGR TRTAIRNGQV WEESLKRLRR DTTLTNVTDP SLDLTALSWE VGCGAPVPLV KCDENSPYRT ITGDCNNRRS PALGAANRAL ARWLPAEYED GLALPFGWTQ RKTRNGFRVP LAREVSNKIV GYLDEEGVLD QNRSLLFMQW GQIVDHDLDF APETELGSNE HSKTQCEEYC IQGDNCFPIM FPKNDPKLKT QGKCMPFFRA GFVCPTPPYQ SLAREQINAV TSFLDASLVY GSEPSLASRL RNLSSPLGLM AVNQEAWDHG LAYLPFNNKK PSPCEFINTT ARVPCFLAGD FRASEQILLA TAHTLLLREH NRLARELKKL NPHWNGEKLY QEARKILGAF IQIITFRDYL PIVLGSEMQK WIPPYQGYNN SVDPRISNVF TFAFRFGHME VPSTVSRLDE NYQPWGPEAE LPLHTLFFNT WRIIKDGGID PLVRGLLAKK SKLMNQDKMV TSELRNKLFQ PTHKIHGFDL AAINLQRCRD HGMPGYNSWR GFCGLSQPKT LKGLQTVLKN KILAKKLMDL YKTPDNIDIW IGGNAEPMVE RGRVGPLLAC LLGRQFQQIR DGDRFWWENP GVFTEKQRDS LQKVSFSRLI CDNTHITKVP LHAFQANNYP HDFVDCSTVD KLDLSPWASR EN RT: 0.00 - 40.04 100

SM: 7G 4.04

NL: 9.92E7 Base Peak MS 031709_lac toperoxidas e_2

31.42

95 90 85 4.13

80 75

4.02

70 Relative Abundance

65 60

25.12

55 25.19

50 45

25.10

40 35

20

3.95

4.22

15 10

1.46

5

3.89

6.47 9.15 4.26 7.79

16.38 15.77

10.61

32.76 32.83

10.71

4.17

25

31.37

10.65

3.99

30

15.74 13.13 14.32

16.41 16.83

24.37 21.53 21.50 22.91 21.33

25.25

31.29

27.71 29.69

32.95 33.92 33.96

0 0

5

10

15

20 Time (min)

25

30

35

35.76 39.53 40

6 Bovine Protein Tryptic Digest Equal Molar Mix Test Certificate (P/N PTD/00001/63) Digest Lot #: 090216TD-63 Each vial contains a total of 100 picomoles of each trypsin digested protein (lyophilized). Dilute to 500ul for a 200 fmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Protein

Qty (pmol)

MW (kD)

Michrom Lot#

Beta Lactoglobulin Lactoperoxidase Carbonic Anhydrase Glutamate Dehydrogenase Alpha Casein Serum Albumin

100 100 100 100 100 100

18.3 79.8 29.2 55.5 23.6 69.3

080318TD-14 080318TD-27 080318TD-11 080318TD-25 080318TD-16 080317TD-15

Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount: MS Source:

Paradigm MS4 – LCQ Deca MS 0.2 x 150mm 3u 200A Magic C18 99.9/0.1 H2O/FA 99.9/0.1 ACN /FA 2.0 ul/min (2000 PSI) 5-35%B in 30 min + hold at 80%B 2 min MS and MS/MS 100 fmol Michrom ADVANCE Source

Chemistry:

Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin)

RT: 0.00 - 40.00 SM: 7G

95

30.93

90

30.99

3.49 3.51

85 80

30.90

75

3.45

70

32.17

20.76 3.56

65 Relative Abundance

NL: 2.21E8 Base Peak MS 022009_6_ equal_2

30.95

100

27.53

60 55

30.11

13.07

50

20.80

3.42

24.79

45 20.69

40 3.60

30

13.03

25

12.83

20

12.03

1.21

15 10

1.28

5

3.65 6.54 5.37

9.61 11.09

15.33

32.35

24.83 27.62

18.95 24.74 21.54 23.91

13.34

32.42

25.19

32.46

27.42

32.60 32.62

7.41 9.22

32.80 33.15

0 0

5

32.28 32.31

27.46

17.07

35

27.57 29.71

10

15

20 Time (min)

25

30

35

37.68

6 Bovine PTD Equal Molar Mix Sequence Information Beta Lactoglobulin: gi|2194089| LIVTQTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP EGDLEILLQK WENGECAQKK IIAEKTKIPA VFKIDALNEN KVLVLDTDYK KYLLFCMENS AEPEQSLVCQ CLVRTPEVDD EALEKFDKAL KALPMHIRLS FNPTQLEEQC HI

Lactoperoxidase: gi|129823| MWVCLQLPVF LASVTLFEVA ASDTIAQAAS TTTISDAVSK VKIQVNKAFL DSRTRLKTTL SSEAPTTQQL SEYFKHAKGR TRTAIRNGQV WEESLKRLRR DTTLTNVTDP SLDLTALSWE VGCGAPVPLV KCDENSPYRT ITGDCNNRRS PALGAANRAL ARWLPAEYED GLALPFGWTQ RKTRNGFRVP LAREVSNKIV GYLDEEGVLD QNRSLLFMQW GQIVDHDLDF APETELGSNE HSKTQCEEYC IQGDNCFPIM FPKNDPKLKT QGKCMPFFRA GFVCPTPPYQ SLAREQINAV TSFLDASLVY GSEPSLASRL RNLSSPLGLM AVNQEAWDHG LAYLPFNNKK PSPCEFINTT ARVPCFLAGD FRASEQILLA TAHTLLLREH NRLARELKKL NPHWNGEKLY QEARKILGAF IQIITFRDYL PIVLGSEMQK WIPPYQGYNN SVDPRISNVF TFAFRFGHME VPSTVSRLDE NYQPWGPEAE LPLHTLFFNT WRIIKDGGID PLVRGLLAKK SKLMNQDKMV TSELRNKLFQ PTHKIHGFDL AAINLQRCRD HGMPGYNSWR GFCGLSQPKT LKGLQTVLKN KILAKKLMDL YKTPDNIDIW IGGNAEPMVE RGRVGPLLAC LLGRQFQQIR DGDRFWWENP GVFTEKQRDS LQKVSFSRLI CDNTHITKVP LHAFQANNYP HDFVDCSTVD KLDLSPWASR EN

Carbonic Anhydrase: IPI00716246 SHHWGYGKHN GPEHWHKDFP IANGERQSPV DIDTKAVVQD PALKPLALVY EATSRRMVN NGHSFNVEYD DSQDKAVLKD GPLTGTYRLV QFHFHWGSSD DQGSEHTVDR KKYAAELHLV HWNTKYGDFG TAAQQPDGLA VVGVFLKVGD ANPALQKVLD ALDSIKTKGK STDFPNFDPG SLLPNVLDYW TYPGSLTTPP LLESVTWIVL KEPISVSSQQ MLKFRTLNFN AEGEPELLML ANWRPAQPLK NRQVRGFPK

Glutamate Dehydrogenase: gi|118533| ADREDDPNFF KMVEGFFDRG ASIVEDKLVE DLKTRQTQEQ KRNRVRGILR IIKPCNHVLS LSFPIRRDDG SWEVIEGYRA QHSHQRTPCK GGIRYSTDVS VDEVKALASL MTYKCAVVDV PFGGAKAGVK INPKNYTDED LEKITRRFTM ELAKKGFIGP GVDVPAPNMS TGEREMSWIA DTYASTIGHY DINAHACVTK PGISQGGIHG RISATGRGVF GHIENFIENA SYMSILGMTP GFGDKTFAVQ GFGNVGLHSM RYLHRFGAKC VAVGESDGSI WNPDGIDPKE LEDFKLQHGT ILGFPKAKIY EGSILEVDCD ILIPAASEKQ LTKSNAPRVK AKIIAEGANG PTTPQADKIF LERNIMVIPD LYLNAGGVTV SYFQILKNLN HVSYGRLTFK YERDSNYHLL MSVQESLERK FGKHGGTIPI VPTAEFQDRI SGASEKDIVH SGLAYTMERS ARQIMRTAMK YNLGLDLRTA AYVNAIEKVF RVYNEAGVTF T

Alpha Casein: IPI00706094 (S1) & IPI00698843 (S2) MK LLILTCLVAVALAR PK HPIK HQGLPQEVLNENLLR FFVAPFPEVFGK EK VNELSK DIGSpESpT EDQAMEDIK QMEAESpISpSpSpEEIVPNSpVEQK HIQK EDVPSER YLGYLEQLLR LK K YK VPQLEIVPNSpAEER LHSMK EGIHAQQK EPMIGVNQELAYFYPELFR QFY QLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEK TTMPLW

Serum Albumin: gi|1351907| MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA

6 Bovine Protein Tryptic Digest Exponential Molar Mix Test Certificate (P/N PTD/00001/64) Digest Lot #: 080318TD-64 See below for amount of each trypsin digested protein (lyophilized) within the vial. Dilute to 500ul for a stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Protein

Qty (fmol)

MW (kD)

Michrom Lot#

Beta Lactoglobulin Lactoperoxidase Carbonic Anhydrase Glutamate Dehydrogenase Alpha Casein Serum Albumin

500,000 50,000 5,000 500 50 5

18.3 79.8 29.2 55.5 23.6 69.3

080318TD-14 080318TD-27 080318TD-11 080318TD-25 080318TD-16 080317TD-15

Paradigm MS4 – LCQ Deca MS Chemistry: 0.2 x 150mm 3u 200A Magic C18 99.9/0.1 H2O/FA 99.9/0.1 ACN /FA 2.0 ul/min (2000 PSI) 5-35%B in 30 min + hold at 80%B 2 min MS and MS/MS 0.02-2000 fmol (2uL of stock standard) Michrom ADVANCE Source 8 SCX fractions (0-1M NaCl) analyzed by RP LC/MS/MS

Q:\Test Mixture QC\2008 Data\expo_mix_1

Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin)

3/20/2008 8:26:01 AM

RT: 0.00 - 40.00 NL: 1.98E10 TIC MS expo_mix_ 1

34.65

100 90

Relative Abundance

80

30.72

70 30.64

60 27.46 50 20.18 40 20.06

30 20 10 1.49 3.58

0 0

2

4

6

14.35 10.12 11.02 14.53 12.33

8

10

12

14

18

34.53

27.40

34.47

31.02

34.73 35.54

34.20 31.24 31.44 34.14

29.15

27.34 20.66 20.77 22.26 24.17 24.38 20.91

16.76 16.96 17.33 16

30.94

28.42

20.43

9.89 9.94 9.55 10.00

6.25 6.31 5.16 5.68 6.49 3.71

20.31

28.12 28.17 28.30

35.71 35.91

32.35

36.10 36.29 37.22

20 Time (min)

22

24

26

28

30

32

34

36

38

40

RT: 0.00 - 40.00 100

NL: 4.05E9 Base Peak MS expo_mix_ 1

30.72

28.17

30.64

90 80 Relative Abundance

Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount: MS Source: 2D Analysis:

28.30

70

30.94

60 50

28.42 31.16

40 20.18 30 9.94 9.62 10.00

6.31

20 10 1.58

0 0

2

3.65

5.16 4

5.60

6

6.36 8.46 8

8.68

14.35 10.92

14.53 14.87

11.15 12.53 10

12

20.06

14

16.60 16.90

16

17.33 18.31 18

31.24 27.46

20.77 20.84 20.91 20.99

20 Time (min)

22

28.54

34.53

30.38 31.44 24.17

29.46

24.38 24.50 27.34 24

26

28

30

32

32.18 34.20 34

34.82

35.71 37.12

36

38

40

6 Bovine PTD Expo Mix Individual Protein Digests RT: 0.00 - 40.04 SM: 7G

22.27

100

Beta Lactoglobulin

50

2.07 2.82 5.09

0 100

25.09 25.64

21.86

35.17 35.41 35.65 38.19 34.58

30.24

34.67

Lactoperoxidase

50 Relative Abundance

24.24

16.19 16.85 7.39 8.86 11.99 12.47 14.00

12.30 13.69

7.29 7.38 1.66 3.34 5.91 7.11

0 100

24.32

18.80 19.06 19.69

27.13

28.25

29.53 30.71

34.45

27.17

Carbonic Anhydrase

50

5.15 7.87 8.03

0.08 2.49

0 100

11.09 9.88

14.35 14.07

Glutamate Dehydrogenase

50

1.69

0 100

8.89 9.05 6.86 8.64

3.50

16.44

16.49

13.72

19.47 20.70

34.78

21.74 23.92

22.65

26.55

27.38

30.42

34.02

35.07 36.07 34.38

34.12

23.73

29.58

25.47

22.54

18.20

36.05

34.70

34.15

21.68

35.00

27.72

29.82

33.20

34.84

35.98 38.24

28.06

Alpha Casein

50

2.18

0 100

27.18

7.50 5.71 7.20 7.82 9.77 12.92 14.15

18.24 19.37

20.46

17.69

Serum Albumin

50

5.95 2.94 3.69 5.03

0 0

5

14.38 13.84 7.89 7.96 12.94 15.40 10

15

19.46

21.94 24.69 30.01 25.26 26.86 24.41 28.06 26.67 22.76 24.54

20 Time (min)

25

33.28 35.28 35.51 39.75

33.75 34.15 35.27 35.61

29.57 30.95 30

35

37.81 40

6 Bovine PTD Expo Mix 2D LC/MS/MS Fractions 1-8 (0-1M

RT: 0.00 - 120.08 100

Fraction 1

50 0 100 50

Fraction 2

0 100

Relative Abundance

50

Fraction 3

0 100 50

Fraction 4

0 100 50

Fraction 5

0 100

Fraction 6

50 0 100

Fraction 7

50 0 100

Fraction 8

50 0 0

10

20

30

40

50

60 Time (min)

70

80

90

100

110

12

6 Bovine PTD Expo Mix Sequence Information Beta Lactoglobulin: gi|2194089| LIVTQTMKGL DIQKVAGTWY SLAMAASDIS LLDAQSAPLR VYVEELKPTP EGDLEILLQK WENGECAQKK IIAEKTKIPA VFKIDALNEN KVLVLDTDYK KYLLFCMENS AEPEQSLVCQ CLVRTPEVDD EALEKFDKAL KALPMHIRLS FNPTQLEEQC HI

Lactoperoxidase: gi|129823| MWVCLQLPVF LASVTLFEVA ASDTIAQAAS TTTISDAVSK VKIQVNKAFL DSRTRLKTTL SSEAPTTQQL SEYFKHAKGR TRTAIRNGQV WEESLKRLRR DTTLTNVTDP SLDLTALSWE VGCGAPVPLV KCDENSPYRT ITGDCNNRRS PALGAANRAL ARWLPAEYED GLALPFGWTQ RKTRNGFRVP LAREVSNKIV GYLDEEGVLD QNRSLLFMQW GQIVDHDLDF APETELGSNE HSKTQCEEYC IQGDNCFPIM FPKNDPKLKT QGKCMPFFRA GFVCPTPPYQ SLAREQINAV TSFLDASLVY GSEPSLASRL RNLSSPLGLM AVNQEAWDHG LAYLPFNNKK PSPCEFINTT ARVPCFLAGD FRASEQILLA TAHTLLLREH NRLARELKKL NPHWNGEKLY QEARKILGAF IQIITFRDYL PIVLGSEMQK WIPPYQGYNN SVDPRISNVF TFAFRFGHME VPSTVSRLDE NYQPWGPEAE LPLHTLFFNT WRIIKDGGID PLVRGLLAKK SKLMNQDKMV TSELRNKLFQ PTHKIHGFDL AAINLQRCRD HGMPGYNSWR GFCGLSQPKT LKGLQTVLKN KILAKKLMDL YKTPDNIDIW IGGNAEPMVE RGRVGPLLAC LLGRQFQQIR DGDRFWWENP GVFTEKQRDS LQKVSFSRLI CDNTHITKVP LHAFQANNYP HDFVDCSTVD KLDLSPWASR EN

Carbonic Anhydrase: IPI00716246 SHHWGYGKHN GPEHWHKDFP IANGERQSPV DIDTKAVVQD PALKPLALVY EATSRRMVN NGHSFNVEYD DSQDKAVLKD GPLTGTYRLV QFHFHWGSSD DQGSEHTVDR KKYAAELHLV HWNTKYGDFG TAAQQPDGLA VVGVFLKVGD ANPALQKVLD ALDSIKTKGK STDFPNFDPG SLLPNVLDYW TYPGSLTTPP LLESVTWIVL KEPISVSSQQ MLKFRTLNFN AEGEPELLML ANWRPAQPLK NRQVRGFPK

Glutamate Dehydrogenase: gi|118533| ADREDDPNFF KMVEGFFDRG ASIVEDKLVE DLKTRQTQEQ KRNRVRGILR IIKPCNHVLS LSFPIRRDDG SWEVIEGYRA QHSHQRTPCK GGIRYSTDVS VDEVKALASL MTYKCAVVDV PFGGAKAGVK INPKNYTDED LEKITRRFTM ELAKKGFIGP GVDVPAPNMS TGEREMSWIA DTYASTIGHY DINAHACVTK PGISQGGIHG RISATGRGVF GHIENFIENA SYMSILGMTP GFGDKTFAVQ GFGNVGLHSM RYLHRFGAKC VAVGESDGSI WNPDGIDPKE LEDFKLQHGT ILGFPKAKIY EGSILEVDCD ILIPAASEKQ LTKSNAPRVK AKIIAEGANG PTTPQADKIF LERNIMVIPD LYLNAGGVTV SYFQILKNLN HVSYGRLTFK YERDSNYHLL MSVQESLERK FGKHGGTIPI VPTAEFQDRI SGASEKDIVH SGLAYTMERS ARQIMRTAMK YNLGLDLRTA AYVNAIEKVF RVYNEAGVTF T

Alpha Casein: IPI00706094 (S1) & IPI00698843 (S2) MK LLILTCLVAVALAR PK HPIK HQGLPQEVLNENLLR FFVAPFPEVFGK EK VNELSK DIGSpESpT EDQAMEDIK QMEAESpISpSpSpEEIVPNSpVEQK HIQK EDVPSER YLGYLEQLLR LK K YK VPQLEIVPNSpAEER LHSMK EGIHAQQK EPMIGVNQELAYFYPELFR QFY QLDAYPSGAWYYVPLGTQYTDAPSFSDIPNPIGSENSEK TTMPLW

Serum Albumin: gi|1351907| MKWVTFISLL LLFSSAYSRG VFRRDTHKSE IAHRFKDLGE EHFKGLVLIA FSQYLQQCPF DEHVKLVNEL TEFAKTCVAD ESHAGCEKSL HTLFGDELCK VASLRETYGD MADCCEKQEP ERNECFLSHK DDSPDLPKLK PDPNTLCDEF KADEKKFWGK YLYEIARRHP YFYAPELLYY ANKYNGVFQE CCQAEDKGAC LLPKIETMRE KVLASSARQR LRCASIQKFG ERALKAWSVA RLSQKFPKAE FVEVTKLVTD LTKVHKECCH GDLLECADDR ADLAKYICDN QDTISSKLKE CCDKPLLEKS HCIAEVEKDA IPENLPPLTA DFAEDKDVCK NYQEAKDAFL GSFLYEYSRR HPEYAVSVLL RLAKEYEATL EECCAKDDPH ACYSTVFDKL KHLVDEPQNL IKQNCDQFEK LGEYGFQNAL IVRYTRKVPQ VSTPTLVEVS RSLGKVGTRC CTKPESERMP CTEDYLSLIL NRLCVLHEKT PVSEKVTKCC TESLVNRRPC FSALTPDETY VPKAFDEKLF TFHADICTLP DTEKQIKKQT ALVELLKHKP KATEEQLKTV MENFVAFVDK CCAADDKEAC FAVEGPKLVV STQTALA

Paradigm LC-MS/MS Certified Digest Test Certificate Equine Cytochrome C (P/N PTD/00001/12) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: MS Source: Amount:

Paradigm MS4 – LCQ Deca XP+ MS 0.2x150mm 3u 200A Magic C18AQ 99.9/0.1% H2O/FA 99.9/0.1% ACN /FA 2.0 ul/min (2200 PSI) 5-35%B in 30 min plus hold 2min at 80%B MS and MS/MS ADVANCE 200 fmol

Source:

Protein MW:

Sigma P/N C-7752 (Horse) Sigma Lot # 077k7001 Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) 11,683 daltons

Digest Lot #: Accession #:

090310TD-12 gi|117995|

Sequence:

GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFTYTD ANKNKGITWK EETLMEYLEN PKKYIPGTKM IFAGIKKKTE REDLIAYLKK ATNE

Chemistry:

RT: 0.00 - 40.04 SM: 7G

NL: 2.56E8 Base Peak MS 031709_Cy to_C_2

18.63

100 95 90 85 80 75

18.61

70 Relative Abundance

65 60 55

18.71

50 45

13.00

3.15

18.56

40

22.31 22.37

3.76

35 3.12

30 25

12.93

20

3.85 3.10 2.68

15 10

1.67

5

6.58 6.27 6.24 6.19

22.44

7.99

13.03 11.38

14.69

18.74

18.40

8.06

18.80

9.08 9.11

19.38

22.45 22.51 27.67 27.33 27.95 29.18 32.80

22.55

0 0

5

10

15

20 Time (min)

25

30

33.71 36.19 35

40

Paradigm LC-MS/MS Certified Digest Test Certificate Equine Apomyoglobin (P/N PTD/00001/04) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount: MS Source: Sequence:

Paradigm MS4 – LCQ Deca MS Source: Sigma P/N A8673 0.2 x 150mm 3u 200A Magic C18AQ Sigma Lot # 087K6018 99.9/0.1 H2O/FA Chemistry: Reduction (Dithiothreitol) 99.9/0.1 ACN /FA Alkylation (Iodoacetic Acid) 2.0 ul/min (2000 PSI) Digestion (TPCK Trypsin) 5-35%B in 20 min + hold at 90%B 2 min Protein MW: 16.9 KD MS and MS/MS Digest Lot #: 080519TD-04 200 fmol Accession #: gi|2554649| ADVANCE Source GLSDGEWQQV LNVWGKVEAD IAGHGQEVLI RLFTGHPETL EKFDKFKHLK TEAEMKASED LKKHGTVVLT ALGGILKKKG HHEAELKPLA QSHATKHKIP IKYNEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFRN DIAAKYKELG FQG

051908_APO_Digest_200fmol_1_080519114737

5/19/2008 11:47:37 AM

RT: 0.00 - 40.01 34.69

90

34.36

80 Relative Abundance

NL: 6.50E9 TIC MS 051908_AP O_Digest_2 00fmol_1_0 805191147 37

34.53

100

34.78

70 60 34.30 33.97

30.98

50 40 30

34.98

20 0.53 1.33

10

3.39 3.96

5.65

7.82 8.67

10.30 11.57 13.73 14.82

18.16

15.56

22.60

19.45 20.87

23.76

26.08

24

26

28.22

29.13

37.15 38.56

31.12

0 0

2

4

6

8

10

12

14

16

18

20

22

28

30

32

34

36

38

40

Time (min) RT: 0.00 - 40.01 SM: 7G NL: 3.78E8

34.35

100

Base Peak MS 051908_APO _Digest_200f mol_1_08051 9114737

90

Relative Abundance

80 70 34.33 60 50 40 34.67

30 18.15 20

18.22

14.84

10

5.36 5.67 5.70

1.40 3.35 3.31

0 0

2

4

6

13.98 14.80

9.02 9.61 10.31 8

10

12

14

17.00

18.27

14.90 16

18

20.85

20 Time (min)

22.60 22.70 22

24

33.97

29.12

26.12 26.02 28.02 29.03 26

28

29.20 30.98 30

32

34.70 34.75 34.78 35.33 37.33

33.90 34

36

38

38.84 40

Paradigm LC-MS/MS Certified Digest Test Certificate Human Alpha Lactalbumin (P/N PTD/00001/40) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at 20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MS4 – LCQ Deca MS 0.2x50mm 3u 200A Magic C18 99.9/0.1% H2O/FA 99.9/0.1% ACN /FA 4.0 ul/min (1200 PSI) 5-35%B in 30 min plus hold 1min @ 90%B MS and MS/MS (with ADVANCE Source) 500 fmole

Sequence:

MRFFVPLFLV GILFPAILAK QFTKCELSQL LKDIDGYGGI ALPELICTMF HTSGYDTQAI VENNESTEYG LFQISNKLWC KSSQVPQSRN ICDISCDKFL DDDITDDIMC AKKILDIKGI DYWLAHKALC TEKLEQWLCE KL

110207_500fmol-halatd_msmsde3_10

Source:

Sigma P/N L-7269 (Human) Sigma Lot # 092K3784 Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) Protein MW: 16,206 daltons Digest Lot #: 071031TD-40 gi|4504947|

Chemistry:

Accession #:

11/5/2007 6:36:25 PM

RT: 0.00 - 39.98 SM: 7G NL: 3.76E7 Base Peak MS 110207_500 fmolhalatd_msm sde3_10

18.18

100 90

Relative Abundance

80 18.11

70 60

18.25

50

13.47 14.09 15.72

40 18.33 30 6.87

20 10

1.76 2.50 1.56

0 0

2

9.53

7.07

5.22 4

6.93

9.59

10.21 12.17

6

8

28.54

16.36

10

20.59 18.41

13.09 12

28.47

20.74

18.83 14

16

18

20 Time (min)

25.93 22.13 23.69 25.34 27.26

22

24

26

33.74 33.91 33.45 34.06 31.85 34.75 35.23 31.16 37.36 39.72

28.62

28

30

32

34

36

38

RT: 0.00 - 39.98 NL: 3.34E9 TIC MS 110207_500 fmolhalatd_ms msde3_10

31.85

100 90

Relative Abundance

80 70 60 50

33.82 33.74

40 30

33.64 33.35

20

18.18

13.47 10 1.06 1.76 0 0

2

6.93

4.45 5.13 4

6

8.41 9.59 9.65 8

10

13.09 12

18.33 20.59 20.74

14.09 15.72 15.85 14

16

18

20 Time (min)

22

25.93 23.61 25.84 24

26.08 26

28.54 28

33.98 34.06 34.16 34.26 34.44 35.33 35.52 37.46

31.16 30

32

34

36

38

39.14

Paradigm LC-MS/MS Certified Digest Test Certificate Human Acid Glycoprotein (P/N PTD/00001/41) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount: MS Source: Sequence:

Paradigm MS4 – LCQ Deca MS Source: Sigma P/N G-9885 (Human) 0.2 x 150mm 3u 200A Magic C18AQ Sigma Lot # 073K7607 99.9/0.1% H2O/FA Chemistry: Reduction (Dithiothreitol) 99.9/0.1% ACN /FA Alkylation (Iodoacetic Acid) 2.0 ul/min (1800 PSI) Digestion (TPCK Trypsin) 5-35%B in 30min + 80%B for 2 min Protein MW: 23.5 kD MS and MS/MS Digest Lot #: 080417TD-41 200 fmol Accession #: gi|112877| Michrom ADVANCE MALSWVLTVL SLLPLLEAQI PLCANLVPVP ITNATLDQIT GKWFYIASAF RNEEYNKSVQ EIQATFFYFT PNKTEDTIFL REYQTRQDQC IYNTTYLNVQ RENGTISRYV GGQEHFAHLL ILRDTKTYML AFDVNDEKNW GLSVYADKPE TTKEQLGEFY EALDCLRIPK SDVVYTDWKK DKCEPLEKQH EKERKQEEGE S

200fmol_acid_glyco_020408_01

5/7/2008 2:04:15 PM

RT: 0.00 - 39.98 SM: 7G NL: 6.56E7 Base Peak MS 200fmol_a cid_glyco_ 020408_01

21.88

100 90

Relative Abundance

32.64

22.91

80 19.93

70 60

34.98

31.68

11.89 25.14

50

35.09 34.82

40

29.99

30

18.31

2.42

9.08

20 2.23 10

2.74 3.10

1.05

7.12

3.49 5.53 6.00

9.19

15.65

9.50 8.58

11.04

8

10

12.98

14.03 14.81

17.54

34.66

27.88

16.52 24.87

20.38

35.35

35.45 29.15

25.82

35.67

27.02

38.55 39.93

0 0

2

4

6

12

14

16

18

20 Time (min)

22

24

26

28

30

32

34

36

38

RT: 0.00 - 39.98 NL: 6.85E9

35.09

100

35.25

90

TIC MS 200fmol_a cid_glyco_ 020408_01

35.01

Relative Abundance

80 70 34.88

60 50

2.42

40

2.38 2.26

30 20

2.22

34.57 34.44 34.41

3.10

3.13

0.19

10

35.49

4.44

32.61 5.32 6.04 7.10

8.66 8.89

11.95 12.78 14.10 14.81

19.96 21.88 22.57 22.91 25.15 17.16 19.23

27.88

29.99

34.34

31.68

32.66 32.74

32

34

35.59 38.20 37.81

0 0

2

4

6

8

10

12

14

16

18

20 Time (min)

22

24

26

28

30

36

38

Paradigm LC-MS/MS Certified Digest Test Certificate Yeast Alcohol Dehydrogenase (P/N PTD/00001/36) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: Amount:

Paradigm MS4 – LCQ Deca MS 0.2 x 50mm 3u 200A Magic C18AQ 99.9/0.1% H2O/FA 99.9/0.1% ACN /FA 4.0 ul/min (1200 PSI) 5-45%B in 20 minutes MS and MS/MS 1 picomole

Sequence:

MSIPETQKGV IFYESHGKLE HKDIPVPKPK ANELLINVKY SGVCHTDLHA WHGDWPLPVK LPLVGGHEGA GVVVGMGENV KGWKIGDYAG IKWLNGSCMA CEYCELGNES NCPHADLSGY THDGSFQQYA TADAVQAAHI PQGTDLAQVA PILCAGITVY KALKSANLMA GHWVAISGAA GGLGSLAVQY AKAMGYRVLG IDGGEGKEEL FRSIGGEVFI DFTKEKDIVG AVLKATDGGA HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQVVKSIS IVGSYVGNRA DTREALDFFA RGLVKSPIKV VGLSTLPEIY EKMEKGQIVG RYVVDTSK

112806_1000fmol-adhtd_4ul-min_msms_02

Source:

Sigma P/N A-7011 (Yeast) Sigma Lot # 041K7480 Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) 36,805 daltons 061201TD-36 gi|1168350|

Chemistry: Protein MW: Digest Lot #: Accession #:

11/29/2006 6:43:24 AM

RT: 0.00 - 30.00 NL: 5.99E8

19.81

100

Bas e Peak F: MS 112806_100 0fm oladhtd_4ulm in_m s m s _ 02

90

Relative Abundance

80 70

17.43 24.67

60 18.12

50

15.92

21.49 19.94

40

21.61 15.44

30 20 7.82 10 1.22 2.80 3.29 3.91

0 0

2

4

6.34 6.94 6

24.77 23.66

11.22

13.82

9.38

10

12

25.65 25.81 26.79 28.73

22.13

8.10 8

25.39

23.56

12.16

14 16 Tim e (m in)

18

20

22

24

26

28

RT: 0.00 - 30.00 NL: 2.45E9 TIC F: MS 112806_10 00fm oladhtd_4ulm in_m s m s _02

24.67

100 90

24.99

Relative Abundance

80 70

25.32 25.49

60 18.51 19.81

50

16.74 17.44

40

16.69 15.36 15.95

30 20 10

0.38 0.84

3.14

5.69 6.34

7.79 7.82 8.74

11.22 11.30 9.38 9.41

25.70

19.89

25.76 23.61

21.49 21.45 23.50 23.46

15.25

25.81 25.92 26.08

29.54

0 0

2

4

6

8

10

12

14 16 Tim e (m in)

18

20

22

24

26

28

Paradigm LC-MS/MS Certified Digest Test Certificate Yeast Enolase (P/N PTD/00001/46) Each vial contains a total of 500 picomoles of trypsin digested protein (lyophilized). Dilute to 500ul for a 1 pmol/ul stock standard (using 2% Acetonitrile/0.005-0.1% ion-pairing acid of choice). Store lyophilized standard at -20oC (shelf life > 6months) and stock solution at 4oC (shelf life >1 month). Instrument: Column: Solvent A: Solvent B: Flow Rate: Gradient: Detection: MS Source: Amount:

Paradigm MS4 – LCQ Deca XP+ MS 0.2x150mm 3u 200A Magic C18AQ 99.9/0.1% H2O/FA 99.9/0.1% ACN /FA 2.0 ul/min (2200 PSI) 5-35%B in 30 min plus hold 2min at 80%B MS and MS/MS ADVANCE 200 fmol

Sequence:

MAVSKVYARS VYDSRGNPTV EVELTTEKGV FRSIVPSGAS TGVHEALEMR DGDKSKWMGK GVLHAVKNVN DVIAPAFVKA NIDVKDQKAV DDFLISLDGT ANKSKLGANA ILGVSLAASR AAAAEKNVPL YKHLADLSKS KTSPYVLPVP FLNVLNGGSH AGGALALQEF MIAPTGAKTF AEALRIGSEV YHNLKSLTKK RYGASAGNVG DEGGVAPNIQ TAEEALDLIV DAIKAAGHDG KVKIGLDCAS SEFFKDGKYD LDFKNPNSDK SKWLTGPQLA DLYHSLMKRY PIVSIEDPFA EDDWEAWSHF FKTAGIQIVA DDLTVTNPKR IATAIEKKAA DALLLKVNQI GTLSESIKAA QDSFAAGWGV MVSHRSGETE DTFIADLVVG LRTGQIKTGA PARSERLAKL NQLLRIEEEL GDNAVFAGEN FHHGDKL

040609_enolaseqc-2

Source:

Protein MW:

Sigma P/N E-6126 (Yeast) Sigma Lot # 109H74501 Reduction (Dithiothreitol) Alkylation (Iodoacetic Acid) Digestion (TPCK Trypsin) 46,784 daltons

Digest Lot #: Accession #:

090403TD-46 gi|119336|

Chemistry:

4/6/2009 8:52:51 AM

RT: 0.00 - 40.07 SM: 7G

NL: 3.88E8 Base Peak MS 040609_en olaseqc-2

32.90

100 95 90 85 80 75

32.88

70

Relative Abundance

65 60 55 24.17

50

24.11

45 40

24.19

35

27.84

16.04 19.97

30 16.03

25

27.16

17.01

11.25

15

16.00 11.18

10 3.90 3.91 5.83 1.58 3.80 7.58

5

10.48

0 0

2

4

6

8

10

15.96 13.45 13.46 13.33 14.01 12

14

21.10 24.07

17.08 19.86 19.83

16

18

32.85

25.05

19.90

11.27

20

27.90

21.93 22.31

20 22 Time (min)

24

27.07 25.08

30.00 31.42 29.98

25.17

29.95 28.03

26

28

32.95 32.98

30

32

34

36.29 36

38.74 38

40