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The Protein Identification Research Group 1999 Sample

Introduction

The purpose of the 1999 ABRF Protein Identification Study was to allow our participating ABRF members to

  1. independently and anonymously evaluate their laboratory’s ability to identify proteins in a digestion mixture at low levels,
  2. provide an introduction and practice to the identification of proteins at low levels for those ABRF laboratories who are not familiar with these techniques,
  3. to evaluate the capability of laboratories to identify a protein by peptide mass searching independent of sample preparation and availability of instrumentation.

The study was designed to eliminate the possibility of sample loss or poor yields due to the use of different digestion and extraction protocols by the participants. Instead, the focus was to determine how well laboratories can identify protein mixtures at low but quantitated level. In order to achieve these goals, the participants were given:

  1. Sample #1: a tryptic digestion mixture of two reduced and with iodoacetamide alkylated proteins with a major component (rHuman Antithrombin III) present at 10 pmoles and a minor component (Bovine Calmodulin) present at 2 pmoles.
  2. Sample #2: a prepared peptide mass data set generated by MALDI MS which has been produced from a separate tryptic protein digest of a Maltose-binding protein:Caldesmon fusion protein (SAMPLE B). To provide assistance for successfully setting up the necessary peptide mass search parameters, a representative reference spectrum from a horse myoglobin tryptic digest (SAMPLE A) was included.

They were asked to identify the two proteins in sample #1 by one or more of the following techniques

  1. MALDI-MS and peptide mass searching
  2. Preparative HPLC/Edman Sequencing
  3. Analytical HPLC/MS and peptide mass searching
  4. Analytical HPLC/MS/MS followed by searching MS/MS fragmentation data.
  5. Other methods as chosen to be appropriate by the participant.

Participants were requested to identify the two protein components in sample #2 specifically by peptide mass searching using the spectrum of horse myoglobin tryptic digest as reference for setting up the search parameters.

Of 109 samples requested, 30 datasets from Sample #1 and 27 datasets from Sample #2 were returned from 31 participating laboratories.

 


 

1999 Protein Identification Research Committee

Roland Annan
UW 2940 — PO Box 1539
SmithKline Beecham Pharm.
709 Swedeland Road
King of Prussia, PA 19406
 
Karl Clauser
Millenium Pharmaceutical Inc.
40 Erie St. FW-1
Cambridge, MA 02139
 
Carol Fiol
Dept. Biochem and Molecular Biology
MRB 355
Colorado State University
Ft. Collins, CO 80523-1870
 
Ulf Hellman
Ludwig Institute for Cancer Research
Box 5959
S-75124 Uppsala
Sweden
 
Kristine Swiderek
ZymoGenetics
2101 Eastlake Ave. E
Seattle, WA 98102
 
Scott D. Patterson
Amgen Inc.
Mammalian Genomics/Proteomics
One Amgen Center Drive, MS 14-2-E
Thousand Oaks, CA 91320-1789

Study Acknowledgements

Amgen Inc.:
Paul Courchesne, Jill Beierle
 
SmithKline Beecham Pharm.:
Drs. Susan Loughery Chen, Francesca
Zappacosta and Xiaolong Zhang

 


 

Overall Summary of Results by Method

ABRF99 Protein Identification Study

 

Method

 

No

 

Major Protein

 

Minor Protein

 

no. called (%)

 

no. called correctly (%)

 

no. called

 

no. called correctly (%)

NanoES

 

2

 

2

 

2

 

0
 

LC-MS/MS

 

5

 

5

 

5

 

2

 

2

Edman

 

8

 

8

 

8

 

3

 

1

Edman/MALDI

 

2

 

2

 

2

 

1

 

0

MALDI

 

11

 

10

 

10

 

6

 

0

LC-MS

 

2

 

2

 

1

 

1

 

0
      

Totals

 

30

 

29 (97%)

 

28 (97%)

 

13 (43%)

 

3 (23%)

77% of the calls for the minor protein were incorrect

 


 

Results Summary from ABRF99 Protein Identification Study

 

Lab

 

Technique

 

Sample Used

 

Major protein identified

 

 

(# of peptides)

 

Minor protein identified

 

 

(# of peptides)

 

Difficulty

 

 

**

 

perform

 

 

protein id

 

 

(success rate)

 

Amount of sample required

99001

 

NanoES

 

40%

 

yes (13)

 

no

 

c

 

yes (80-90%)

 

0.5 pmol

99002

 

MALDI

 

40%

 

yes (4)

 

no

 

c

 

yes (50%)

 

10 pmol

99003

 

Edman

 

100%

 

yes (3)

 

no

 

c

 

yes (99%)

 

10 pmol

99004

 

MALDI

 

5%

 

yes (13)

 

no

 

c

 

yes (90%)

 

0.1-5 pmol

99005

 

MALDI

 

?

 

no

 

no

 

tc/vc

 

yes (80%)

 

10 pmol

99006

 

Edman

 

100%

 

yes (5)

 

no

 

c

 

yes (90%)

 

5 pmol

99007

 

MALDI

 

10%

 

yes (9)

 

no

 

c

 

no

 

5-10 pmol

99008

 

LC MS/MS

 

5%

 

yes (19)

 

yes (5)

 

e

 

yes (90%)

 

1 pmol

99009

 

LC MS/MS

 

40%

 

yes (4)

 

no

 

vc

 

?

 

>1 pmol

99009

 

MALDI

 

5%

 

yes (11)

 

no

 

c

 

?

 

1 pmol

99010

 

LC MS

 

100%

 

no

 

no

 

c

 

yes (30-50%)

 

50-100 pmol

99011

 

LC MS/MS

 

2%

 

yes (21)

 

yes (5)

 

e

 

yes (95%)

 

1 pmol

99012

 

MALDI

 

10%

 

yes (13)

 

no

 

e/vc

 

yes (90%)

 

1-2 pmol

99013

 

LC MS

 

10%

 

yes (13)

 

no

 

vc

 

yes (50%)

 

2-5 pmol

99014

 

MALDI

 

2%

 

yes (3)

 

no

 

c

 

yes (90%)

 

1-2 pmol

99015

 

Edman

 

100%

 

yes (1)

 

no

 

c

 

yes (50%)

 

10 pmol

99017

 

Edman

 

100%

 

yes (2)

 

no

 

c

 

yes (80%)

 

10 pmol

99018

 

Edman

 

80%

 

yes (3)

 

no

 

vc

 

yes (60%)

 

20 pmol

99019

 

Edman

 

90%

 

yes (4)

 

no

 

vc

 

?

 

5-50 pmol

99020

 

MALDI

 

50%

 

yes (19)

 

no

 

c

 

?

 

?

99021

 

LC MS/MS

 

1%

 

no*

 

no*

 

c

 

yes (95%)

 

0.5 pmol

99022

 

MALDI

 

?

 

yes (17)

 

no

 

e

 

yes (60%)

 

2-10 pmol

99023

 

Edman

 

100%

 

yes (17)

 

yes (2)

 

e/c

 

yes (75%)

 

1 pmol

99024

 

LC MS/MS

 

100%

 

yes (9)

 

no

 

c

 

yes (90%)

 

10 pmol

99025

 

Edman/MS

 

90%

 

yes (4)

 

no

 

vc

 

yes (90%)

 

10 pmol

99026

 

Edman

 

66%

 

yes (3)

 

no

 

vc

 

yes (100%)

 

10-20 pmol

99027

 

MALDI

 

5%

 

yes (7)

 

no

 

c/tc

 

yes (60%)

 

10 pmol

99028

 

MALDI

 

10%

 

yes (6)

 

no

 

c

 

yes (90%)

 

2 pmol

99029

 

NanoES

 

70%

 

yes (8)

 

no

 

c

 

?

 

1-10 pmol

99030

 

Edman/MS

 

90%

 

no

 

no

 

vc

 

?

 

10-25 pmol

* Instrument failure

** e = easy c = challenging vc = very challenging tc = too challenging

 


 

Summary of Edman Sequencing Results for ABRF99 Protein Identification Study

 

Lab

Column size, type

 

Chromatogram quality
(1-5, 5 best)

 

peptides sequenced

 

peptides

 

 

yielding

 

 

sequence

 

pmol yield 2ndresidue

 

major protein identified
(no of peptides)

 

minor protein identified
(no of peptides)

Time spent*

 

99003

2.0x250 mm, C18

 

2

 

3

 

3

 

0.8

 

Yes (3)

 

No

 

70 h

 

99006

1.0x250 mm, C8

 

4

 

7

 

5

 

1.1

 

Yes (5)

 

No

 

5 h

 

99015

1.0x125 mm, C18

 

2

 

2

 

2

 

1.9

 

Yes (1)

 

No

 

13 h

 

99017

2.1x100 mm, C2/C18

 

4

 

3

 

2

 

1.4

 

Yes (2)

 

No

 

4 h

 

99018

1.0x100 mm, C18

 

2

 

6

 

4

 

0.8

 

Yes (3)

 

No

 

24 h

 

99019

2.1x30 mm, C18

 

3

 

8

 

7

 

1.8

 

Yes (4)

 

No

 

Too much

 

99023

0.8x150 mm, C18

 

5+

 

21

 

19

 

1.4

 

Yes (17)

 

Yes (2)

 

10 days (!)

 

99025

1.0x250 mm, C18

 

5

 

5

 

5

 

2.0

 

Yes (4)

 

No

 

40 h

 

99026

0.5x150 mm, C18

 

3

 

3

 

3

 

1.4

 

Yes (3)

 

No

 

10 h

 

99030

1.0x250 mm, C18

 

3

 

1

 

0

 

-

 

No

 

No

 

5 h

* Some labs included sequencing time in their estimate and some labs did not.

 


 

HPLC Purification of Tryptic Peptides from ABRF99 Protein Identification Study.

Lab: 99023 Column: 0.8mm x 150mm C18

 


 

Anti Thrombin Coverage Map by Edman Sequencing from 99023

HGSPVDICTA KPRDIPMNPM CIYRSPEKKA TEDEGSEQKI PEATNRRVWE 50

LSKANSRFAT TFYQHLADSK NDNDNIFLSP LSISTAFAMT KLGACNDTLQ 100

QLMEVFKFDT ISEKTSDQIH FFFAKLNCRL YRKANKSSKL VSANRLFGDK 150

SLTFNETYQD ISELVYGAKL QPLDFKENAE QSRAAINKWV SNKTEGRITD 200

VIPSEAINEL TVLVLVNTIY FKGLWKSKFS PENTRKELFY KADGESCSAS 250

MMYQEGKFRY RRVAEGTQVL ELPFKGDDIT MVLILPKPEK SLAKVEKELT 300

PEVLQEWLDE LEEMMLVVHM PRFRIEDGFS LKEQLQDMGL VDLFSPEKSK 350

LPGIVAEGRD DLYVSDAFHK AFLEVNEEGS EAAASTAVVI AGRSLNPNRV 400

TFKANRPFLV FIREVPLNTI IFMGRVANPC VK 432

 

Note: The colors on the coverage maps indicate sequences covered. Red and blue are used to separate adjacent tryptic peptides.

 


 

Summary of Calmodulin Coverage

LC-MS/MS or Edman

 

mass

 

residues

 

99008

 

 

LC MS/MS

 

99011

 

 

LC MS/MS

 

99023

 

 

Edman

 

PIRC-1

 

 

LC MS/MS

 

PIRC-2

 

 

LC MS/MS

 

907

 

22-30

 

+

 

+
 

 

+
 

 

956

 

14-21

 

+

 

+
 

 

+
 

 

1093

 

78-86

 

+
    

 

1265

 

95-106
 

 

+

 

+
  

 

1563

 

1-13
 

 

+
 

 

+
 

 

1596

 

78-90

 

+
 

 

+
  

 

2086

 

78-94

 

+
    

 

2521

 

127-148*
 

 

+
   

* Contains two oxidized methionines

 


 

Summary of MALDI-TOF Results From ABRF99 Protein Identification Study

 

 Lab

 

Amount sample used (%)

 

 De-salted

 

Total Peaks

 

 Major protein peaks

 

 Minor protein peaks

 

 MS Calib.Method

 

 Search Engine

 

 Mass Tol. (+/-)

 

 Time Spent (Hrs)

 

 Level* Of Difficulty

 

Lab Does Routine Protein ID

 

NormalSample Needed (pmol)

99001

 

40

 

-

 

16

 

13

 

-

 

external

 

MS-Fit

0.2 Da

 

8

 

C

 

yes

 

0.5

99002

 

40

 

-

 

32

 

4

 

-

 

internal

 

MS-Fit

 

 

GPMAW

30 ppm

 

3

 

C

 

yes

 

10

99004

 

5

 

-

 

57

 

13

 

-

 

external

 

MS-Fit PeptIdent

100 ppm

 

5-10

 

C

 

yes

 

0.1-5

99005

 

-

 

-

 

-

 

-

 

-

 

external

 

-

-

 

-

 

TC

 

yes

 

10

99007

 

10

 

-

 

27

 

9

 

-

 

internal

 

Profound

 

 

Peptide Search

0.1 Da

 

12

 

C

 

no

 

5-10

99009

 

40

 

-

 

16

 

8

 

-

 

internal
 

40 ppm

 

6

 

C

 

yes

 

1

99012

 

10

 

-

 

33

 

13

 

-

 

external

 

GPMAW

200 ppm

 

2

 

C

 

yes

 

1-2

99014

 

5

 

RP guard

 

9

 

3

 

-

 

external

 

MS-Fit

 

 

MS-Tag

500 ppm

 

8

 

C

 

yes

 

1-2

99020

 

50

 

ZipTip

 

48

 

19

 

-

 

external

 

MS-Fit

100 ppm

 

 

C

 

no

 

-

99022

 

5

 

ZipTip

 

35

 

17

 

-

 

external

 

MS-Fit

0.05%

 

8

 

E

 

yes

 

-

99025

 

2

 

-

 

22

 

8

 

-

 

none

 

Profound

0.5 Da

 

40

 

VC

 

yes

 

10

99027

 

2

 

-

 

16

 

7

 

-

 

internal

 

PeptIdent

100 ppm

 

4

 

C

 

yes

 

10

99028

 

10

 

-

 

14

 

6

 

-

 

internal

 

MS-Fit

100 ppm

 

4

 

C

 

yes

 

2

99030

 

10

 

-

 

-

 

10

 

-

 

internal

 

Profound

 

 

Peptide Search

0.5 Da

 

5

 

VC

 

yes

 

10-25

* TC — too challenging, VC — very challenging, C - challenging, E — easy.

 


 

Summary of Protein Identification from Paper Sample

Lab

Protein 1

Protein 2

Search Engine

Sample A**

99001

Maltose Binding Protein

Caldesmon

MS FIT

MALDI/NanoES

99002

Myosin

Caldesmon

MS FIT

MALDI

99004

Maltose Binding Protein

Caldesmon

MS FIT

MALDI

99005

no call *

Caldesmon

MS FIT

MALDI

99006

Myosin

Caldesmon

Peptide Search

Edman

99007

Maltose Binding Protein

Caldesmon

Peptide Search

MALDI

99008

Myosin

Caldesmon

MS FIT

LC-MS/MS

99010

N12_STRPY

Caldesmon

ProFound

LC-MS

99011

Myosin

Spectrin

MS FIT

LC-MS/MS

99012

Maltose Binding Protein

Caldesmon

GPMAWs

MALDI

99013

Myosin

Plectin

Peptide Search

LC-MS

99014

S. cerevisiae ORF

Caldesmon

ProFound

MALDI

99015

Myosin

Caldesmon

ProFound

Edman

99016

Plectin

Dystrophin

PepIdent

 

99017

Myosin

Caldesmon

MS FIT

Edman

99018

Plectin

Caldesmon

PepIdent

Edman

99019

Myosin

Caldesmon

MS FIT

Edman

99020

Maltose Binding Protein

Caldesmon

MS FIT

MALDI

99021

Maltose Binding Protein

Caldesmon

ProFound

LC-MS/MS

99022

S. pombe ORF

C. elegans ORF

MS FIT

MALDI

99024

Myosin

Caldesmon

MS FIT

LC-MS/MS

99025

Guanylate Transferase

Caldesmon

ProFound

Edman/MALDI

99026

Maltose Binding Protein

Caldesmon

PepIdent

Edman

99027

no call

Caldesmon

PepIdent

MALDI

99028

Myosin

Caldesmon

MS FIT

MALDI

99029

Maltose Binding Protein

Caldesmon

Peptide Search

NanoES

99030

Guanylate Transferase

Caldesmon

ProFound

Edman/MALDI

Maltose Binding Protein was listed on the output list

** Technique used to identify proteins in the "live" sample

 


 

Summary of LC-MS/MS Results for ABRF99 Protein Identification Study

Laboratory

column

diameter

sample injected

precursors sequenced

precursors yielding useful spectra

major protein identified

(no of peptides)

minor protein identified

(no of peptides)

reason for not finding minor component

hours spent working on study

99008

0.075 mm

5%

139

29

yes (19)

yes (5)

 

3

99009

0.100 mm

40%

4

4

yes (4)

no

sensitivity

8

99011

0.075 mm

2%

31

29

yes (21)

yes (5)

 

2

99021

0.300 mm

1%**

3

0

no

no

problems

8

99024

1.00 mm

100%

23

9

yes (9)

no

sensitivity

4

PIRC-1***

0.180 mm

60%*

39

32

yes (21)

yes (3)

 

8

PIRC-1***

0.075 mm

10%

  

yes (?)

yes (?)

  

* The flow was split 10:1 after the column, so 6% of the total sample was consumed, the remainder was fraction collected

** The entire sample was lost in an injector failure. A rinse of the tube was used for a second analysis

*** Protein Identification Research Committee (PIRC) laboratory

 


 

Summary of Anti Thrombin Coverage by LC-MS/MS

mass

residues

99011

99008

99009

99024

99021

PIRC-1

PIRC-2

579

130-133

+

  

+

   

659

140-145

+

  

+

   

699

237-241

+

  

+

   

761

48-53

+

      

799

40-46

     

+

 

832

177-183

 

+

     

839

108-114

+

  

+

 

+

 

850

229-235

     

+

 

860

170-176

+

  

+

 

+

 

908

325-332

+

    

+

 

917

47-53

+

+

 

+

 

+

 

978

126-132

+

  

+

 

+

 

994

229-236

 

+

     

1093

227-236

 

+

     

1211

323-232

+

+

   

+

 

1221

29-39

 

+

     

1233

404-413

+

+

+

  

+

 

1310

360-370

+

      

1340

115-125

+

+

+

  

+

 

1381

1-13*

 

+

   

+

 

1389

414-425

+

      

1430

263-275

+

+

+

  

+

 

1437

1-13

+

    

+

 

1528

58-70

+

+

+

  

+

 

1675

170-183

+

+

 

+

 

+

 

1875

30-46

+

+

   

+

 

2031

30-47

 

+

 

+

   

2291

371-393

+

      

2359

71-91*

     

+

 

2417

349-370

+

      

Percent Coverage

46%

38%

11%

18%

 

40%

 

 


 

Anti Thrombin Coverage Map by LC-MS/MS from 99011

 

 

HGSPVDICTA KPRDIPMNPM CIYRSPEKKA TEDEGSEQKI PEATNRRVWE 50

LSKANSRFAT TFYQHLADSK NDNDNIFLSP LSISTAFAMT KLGACNDTLQ 100

QLMEVFKFDT ISEKTSDQIH FFFAKLNCRL YRKANKSSKL VSANRLFGDK 150

SLTFNETYQD ISELVYGAKL QPLDFKENAE QSRAAINKWV SNKTEGRITD 200

VIPSEAINEL TVLVLVNTIY FKGLWKSKFS PENTRKELFY KADGESCSAS 250

MMYQEGKFRY RRVAEGTQVL ELPFKGDDIT MVLILPKPEK SLAKVEKELT 300

PEVLQEWLDE LEEMMLVVHM PRFRIEDGFS LKEQLQDMGL VDLFSPEKSK 350

LPGIVAEGRD DLYVSDAFHK AFLEVNEEGS EAAASTAVVI AGRSLNPNRV 400

TFKANRPFLV FIREVPLNTI IFMGRVANPC VK 432

 

Note: The colors on the coverage maps indicate sequences covered. Red and blue are used to separate adjacent tryptic peptides.

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