Protein Identification ABRF99 Poster

The Protein Identification Research Group 1999 Sample

Introduction
Overall Summary of Results by Method
Results Summary
Summary of Edman Sequencing Results
HPLC Purification of Tryptic Peptides from ABRF99 Protein Identification Study. Lab: 99023
Anti Thrombin Coverage Map by Edman Sequencing from 99023
Summary of Calmodulin Coverage LC-MS/MS or Edman
Summary of MALDI-TOF Results
Summary of Protein Identification from Paper Sample
Summary of LC-MS/MS Results
Summary of Anti Thrombin Coverage by LC-MS/MS
Anti Thrombin Coverage Map by LC-MS/MS from 99011

Introduction

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

independently and anonymously evaluate their laboratory’s ability to identify proteins in a digestion mixture at low levels,
provide an introduction and practice to the identification of proteins at low levels for those ABRF laboratories who are not familiar with these techniques,
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:

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.
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

MALDI-MS and peptide mass searching
Preparative HPLC/Edman Sequencing
Analytical HPLC/MS and peptide mass searching
Analytical HPLC/MS/MS followed by searching MS/MS fragmentation data.
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 2^nd residue
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