Protein Sequencing Research Group: Results of the PSRG 2012 Study Terminal Sequencing of Standard Proteins in a Mixture Year 1 of the 2-year Study.

Protein Sequencing Research Group: Results of the PSRG 2012 Study

Terminal Sequencing of Standard Proteins in a Mixture

Year 1 of the 2-year Study

Current PSRG Members

Henriette Remmer (Co-Chair) University of Michigan Jim Walters (Co-Chair) Sigma-Aldrich Robert English* University of Texas Medical Branch Pegah Jalili* Sigma-Aldrich Viswanatham Katta Genentech, Inc Kwasi Mawuenyega Washington University School of Medicine Detlev Suckau Bruker Daltonics Bosong Xiang Monsanto, Co. Jack Simpson (EB liaison) United States Pharmacopeia

* new members added in 2011

PSRG 2012/13 – Study Background and Design

Status of Terminal Sequencing : In the midst of a technology transition from classical Edman sequencing to mass

spectrometry (MS) based sequencing Both technique have varied strengths and weaknesses and both have a role in

biochemical research. With a complimentary role realized, we attempt to push the capabilities of the various

sequencing techniques, namely terminal sequencing of proteins in mixture

Concept of the 2012 Study- Terminal Sequencing of Proteins in a Mixture: Sequencing proteins in a mixture requires separation of proteins prior to analysis

Edman Sequencing : SDS-PAGE and electroblotting prior to analysis – well established in most core facilities MS based sequencing: LC separation necessary prior to analysis- not well established in most core facilities

=> PSRG designed a 2-year studyYEAR 1: Terminal sequencing and identification

of three separated standard proteins YEAR 2: Same three proteins distributed, this time in mixture

PSRG 2012 Year 1: Study Objective

To obtain N-terminal sequence information on three standard proteins supplied as

separated samples.

2011 Study Design – The Samples

Participants were asked to analyze the samples for terminal sequencing using any technology available

Participants obtained all three proteins with ID in sufficient amounts to sequence each protein utilizing all three technologies. Feasibility of analysis had been validated by PSRG members.

Participants also filled out a survey, all responses were kept anonymously

Protein Name

Amounts Provided (pmol)

N-terminally blocked?

Fusion Protein?

Comments

BSA 1mg No No reference protein/ calibrant

Protein A 3x 100 Yes Yes Fusion protein with blocked N-terminus

Endostatin 3x 100 No No Contains two N-terminal variants

Participation and Survey results 25 laboratories from 12 countries requested samples for Edman sequencing and

most of the labs (23) also for MS sequencing.

14 of the 25 participating laboratories (56%) completed the survey.

7 of the 14 labs utilized Edman sequencing , 6 top-down MS and 6 bottom-up MS.

Out of 14 respondents, 9 labs analyzed the reference protein BSA, 8 correctly determined the N-terminus 13 labs analyzed Protein A , 5 correctly determined the N-terminus 14 labs analyzed Endostatin, 12 labs correctly determined the N-terminus , only 7

identified the presence of the second N-terminus

BSA Protein A Endostatin02468

10121416

Participation and Survey ResultsCorrectly deter-mined the N-ter-minusUnable to de-termine the N-terminus

Nu

mb

er

of

Re

-s

po

nd

en

ts

Survey Response Results

Top -d

own

mas

s sp

ectro

met

ry a

nalys

is

Botto

m-u

p m

ass

spec

trom

etry

ana

lysis

Edman

Deg

rada

tion

In S

ourc

e Dec

ay

T3 Seq

uenc

ing

ECD/ETD

Tande

m M

S

Inta

ct M

W M

easu

rem

ent

Electro

elutio

n

Bioche

mica

l Lab

eling

or C

aptu

re0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

What types of analyses did you perform on the sample?

Purification and separation method before analysis

N-Terminal Techniques:Edman Degradation

Edman Workflows

PSRG 2012 Samples

Used sample as Provided

(5)

ABI Procise 4 - 494 HT’s 1 – 492 cLC 2 - 494 cLC

SDS PAGE – blotting on PVDF

(2)

blotting on PVDF (1)

Shimadzu PPSQ-33A

Edman sequencing Protein A

PROTEIN A- FUSION PROTEIN- N-TERMINUS BLOCKED

C10

Polybrene-precycled glass fiber filters

ABI Procise Biosystems Model 494HT

De-blocking(PGAP)

100 pmol

Sequence 1 MMet L R P V E T P

C10 - L R P V E T P

Edman sequencing of Endostatin A00

Probability 2: position 7 Histine to Glutamine

blotting on PVDF Shimadzu PPSQ-33A

H2O with 0.1 % TFA

Probability 1: position 4 Proline to Arginine

Initial Yield: 36.95 %Repetitive Yield: 84.98 %

Edman sequencing of Endostatin A00

Sequence 1 D F Q P V L H L V A L N S P L

A00/Vaiants 1 D F Q P V L H L V A L N S P L

Sequence 2 H S H R D F Q P V L H L V A L

A00/Variant 2 R Q

Sequence Verification:

with Blast P

Information about the sequence:

SwissProt output

Summary of N-terminal sequencing result

Sample Description

Lab ID Amino acid sequence

BSA Y20 D T H K S E I A H R F K D L G E E H F K G L V L I A F S Q Y L Q Q X P F D E H V K L V N

C10 D T H K S E I A H R F K D L G E E H F K G L V L I A F S Q Y

N32 D T H K S E I A H R F K D L G E E H F K G L V L I

A00 D T H K S E I A H R F K D L G E E H F K G L V L I A F S Q Y

Protein A Y20 F L R P V E T P T R E I K K L D G L A Q H D E A Q Q N A F Y Q V L N M P N

Y20 M F L R P V E T P T

C10 L R P V E T P T R E I K K L D G L A Q H D E A Q Q N A F Y Q V L

N32 X L R P V E T P X R E I K K L

A00 M L R P V E T P T R E I K K L D G L

S10 X L R P V E T P T R E I K K L D G L A Q H D E A Q Q N A

V00 F L R P V E T P T R E I K K L D G L A Q H D E A Q Q N A F Y Q V L N M P N

Endostatin Seq. 1 Y20 D F Q P V L H L V A L N S P L S G G M R G I R G A D F Q X F Q Q A

C10 D F Q P V L H L V A L N S P L S G G M R G I R G A D F Q C F Q Q A R

E20 D F Q P V L H L V A L N S P L S G G M R G I R G A D F Q C F Q Q A R A V G L A G T

N32 D F Q P V L H L V A L N S P L S G G M R G I

A00 D F Q P V L H L V A L N S P L

S10 D F Q P V L H L V A L N S P L S G G M R G

Endostatin Seq. 2 Y20 H S H R D F Q P

C10 H S H R D F Q P X L H X X A L N X X X S G G M

E20 H S H R D F Q P V L H L V A L N S P L S G G M R G I R G A D F Q C

N32 H S H R D F Q P V X H X V A L N S

PSRG 2011 Edman Conclusions & Observations

All lab returned N-terminal data which correlate well with the published protein sequences

It can produce the data with and without separation (SDS PAGE and chromatography)

No C-terminal data was produced with Edman.

If the protein N-terminally blocked, the reaction will not proceed for most but not all modifications.

The reagents for Edman sequencing are very expensive

Edman sequencing allows for direct determination of

the protein’s N-terminal sequence.

N-Terminal Techniques Overview: MS Techniques

Mass Spectrometry Methods Used

Top-Down Sequencing (no digests) ISD, T³: AB Sciex 4800

MALDI-TOF/TOF MS, ISD, T³: Bruker Ultraflex

MALDI-TOF/TOF MS, ETD,CID: Bruker maXis 4G UHR-QTOF

Only Top-Down N-term results were returned.

Some participants used Bottom-Up MS as validation step

Bottom-Up MS/MS (digests) MALDI-TOF/TOFs: AB/Bruker ESI-Orbitrap: Thermo

Top-Down Experimental

Bruker UltraflexBruker UltrafleXtreme

HPLC

Direct infusion

As provided

SampleSeparation

Top-Down Instrumentation

0.1% TFAMeOH/H2O/HOAc

6M GndHClVarious organic/H2O/acid

AB Sciex 4800

Triversa NanomateAgilent 1200

Bruker Autoflex speed

Bruker MaXis 4G

ISD/T³

ISD/T³

ISD

ETDCID

Software used for MS Top-Down Analysis

BioTools 3.2: Sequence-tags, automatic de-novo sequencing, trigger Mascot TD searching, result visualization, terminal assignments, TD report generation(Bruker)

Mascot 2.3: TD and BU Database searches (Matrix Science)

BLAST/MS-BLAST: Protein identification based on sequence tags(NIH, Harvard/EMBL)

ISDetect: Sequence-tags, semi-automatic de-novo sequencing, result visualization (Genentech, Y Gan et al, in prep. )

The Top-Down MS Standard Analysis Strategies

MW Determination: Check Sample Quality + Final QC ETD/ISD: obtain internal sequence Tags

ID Protein: e.g. Mascot search Extend Sequence towards N-terminus (and C-term alike)

Compare with obtained protein sequences incl. PTMs) T³-Sequencing, i.e. MS/MS analysis of MALDI-ISD fragments Edman sequencing

Problems: unknown terminal modifications (Sample B), fusion proteins (Sample B), ragged ends (Sample C)

D T H K S E I A H R F K D L G E E H F K G L V L I A F S Q Y L Q Q C P

D T H K S E I A H R F K D L G E E H F K G L V L I A F S Q Y L Q Q C P

BSA ISD Spectrum in DAN matrix PSRG123good calibrant for ISD Spectra

Sample A: BSA, ISD+Edman C10following the basic strategy

BSA sequence Accession number: AAI02743 c-ions in the MALDI-ISD spectrum revealed the sequence from

Arg10 -Tyr30. Edman sequencing provided Asp1 to Gly15 Data from the orthogonal methods were put together to obtain 30

residues of BSA sequence.

FINAL SEQUENCE OBTAINED FOR BSA: 1 10 20 30 40

DTHKSEIAH RFKDLGEEHF KGLVLIAFSQ YLQQCPFDEH VKLVNELTEF…

Coverage by Edman

Coverage by MALDI-ISD

Coverage by both

Sample B Endostatin (donated by Sigma)issues: ragged N-term, C-term loss of K

C-term K excised

C-term K excised

added

Endostatin L36Annotated ISD Spectrum from on/off gradient

Interferingcomponent

Endostatin L36HPLC chromatogram, separation of two variant, ISD of F1, F2 not assigned

The recovery from the endostation samplemight be lower than 100 pmol

2x10

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

VWD1 - A:Absorbance Sig=214 Endostatin.d

Response vs. Acquisition Time (min)0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6

100 pmol Myoglobin standard F1

F2

LC-separation detected the protein heterogeneity, removed polymeric contaminationbut reduced the sample amount and readout length

UHR-QTOF MS analysis of Endostatin: 2 Components

1221.9913

1297.3352

1390.0011

1496.8469

1621.4171

1768.8184

1945.6003

+MS,

0.0

0.2

0.4

0.6

0.8

1.0

5x10Intens.

1200 1400 1600 1800 2000 m/z

Z10

In contrast to MALDI-ISD, the QTOF-ETD analysis takes place after precursor ion selection

ETD Analysis of Endostatin, First Precursor: Mascot Database Search Result

Simplest Use of Top-Down Data: Mascot Search

Z10

TDS Analysis of Endostatin, First Precursor: Deconvoluted and Annotated ETD Spectrum

c 2 c 9 c 26

Z10

TDS Analysis of Endostatin, First Precursor: Mass Accuracy of intact Protein

+MS, 0.5-20.4min, Deconvoluted (MaxEnt)

19436.82291+

19437.82561+

19438.82821+

19439.83081+

19440.83341+

19441.83591+

19442.83831+

19443.84081+

19444.84321+

19446.84791+

19447.85021+

19448.85261+

19449.85481+

19450.85711+

19451.85941+

19452.86161+

19453.86381+

C866H1340N250O250S6, 19433.8151

0

1

2

3

4

5x10Intens.

19436 19438 19440 19442 19444 19446 19448 19450 19452 19454m/z

Measured Monoisotopic mass 19433.8783Theoretical Monoisotopic mass 19433.8151

Mass error 3.2 ppm

Measured (black) Spectrum Simulated (red) Spectrum

Z10

Precision MW allows to confirm proper N-term and C-term loss of Lysin

Endostatin: TDS Sequence 1 PSRG123

Endostatin: TDS Sequence 2 PSRG123

If ISD spectral quality is good, both sequences can be directly read and N- and C-termini can be assigned from THE SAME SPECTRUM

Rec. Protein A (donated by Repligen)Issues: N-term methylation, fusion site after residue 18

E.coli b-Glucuronidase

SPA_STAAU C-term sequence does not match intact MW(nice challenge for Top-Down MS in the Future..)

2340. 0 2875. 2 3410. 4 3945. 6 4480. 8 5016. 0

Mass (m/ z)

789. 6

0

10

20

30

40

50

60

70

80

90

100

% Inten

sity

4 7 0 0 Re fle c tor Spe c # 1 MC=>BC=>SM5 [BP = 1 05 6 .5 , 9 64 0 ]

2445.186

2560.194

3016.333

2689.229

2410.025

2760.251

2888.299

3130.361

3201.401

3512.514

3348.530

3965.857

4308.395

3851.577

4606.874

4420.955

3738.646

4193.676

4705.008

4535.005

4848.976

899. 0 1189. 2 1479. 4 1769. 6 2059. 8 2350. 0

Mass (m/ z)

9. 6E+3

0

10

20

30

40

50

60

70

80

90

100

% Inten

sity

4 7 0 0 Re fle c tor Spe c # 1 MC=>BC=>SM5 [BP = 1 05 6 .5 , 9 64 0 ]

1056.538

955.500

1212.624

919.333

934.331

1823.966

957.500

1058.383

1454.726

1341.656

1042.506

1582.809

903.327

971.467

1710.888

1188.525

1013.273

998.272

1470.682

1938.978

1087.428

1995.994

1073.412

1215.572

1756.740

2180.096

2308.141

944.343

1161.564

1245.541

2109.059

1740.760

1027.501

984.284

926.387

1641.726

1357.615

1105.392

1137.325

1885.778

1203.549

2295.997

1626.733

1419.635

1656.733

2013.854

1228.526

1260.549

1401.607

1289.606

1797.780

1547.683

1327.631

1809.931

1440.693

1304.601

1485.682

1568.760

1774.780

1912.813

1599.729

1120.359

2070.882

1724.774

1383.634

2041.839

1370.625

2029.874

1835.913

ISD Spectrum Protein A (DAN) E20manual sequence generation

T R E I/L K/Q K/Q I/L DG I/L K/QA

H D

E A K/QK/Q

ISD spectrum for Samples #2 (Protein A) was manually interpreted by sequential subtraction of ionsResultant sequence:

was Blasted against the Dayhoff public database (below)

Protein A Identification E20

TRE[IL][KQ][KQ][IL]DG[IL]A[KQ]

Only two sequences matched.

Homology searching of the N-term Tag provided a) b-Glucuronidase, b) its N-terminallyextended sequence, c) mass offset indicates N-term Methylation

Protein A MS/MS E20ISD c-ion m/z 1056.538

T³-sequence analysis of c9 confirms N-term methylation

Protein A L36MS/MS of N-terminal tryptic fragment

M L R P V E T P T R Met Leu Arg Pro Val Glu Thr Pro Thr Arg

Ion 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10

a M* L R P V E T P T R 118.068 231.153 387.254 484.306 583.375 712.417 813.465 910.518 1011.566 1167.667

b M* L R P V E T P T R 146.063 259.147 415.249 512.301 611.370 740.412 841.460 938.513 1039.560 1195.662

a-17 M* L R P V E T P T R 101.042 214.126 370.227 467.280 566.348 695.391 796.439 893.491 994.539 1150.640

b-17 M* L R P V E T P T R 129.037 242.121 398.222 495.275 594.343 723.386 824.433 921.486 1022.534 1178.635

y M* L R P V E T P T R 175.119 276.167 373.219 474.267 603.310 702.378 799.431 955.532 1068.616 1213.672

i M* L R P V E T P T R 118.068 86.096 129.113 70.065 72.081 102.055 74.060 70.065 74.060 129.113

10 9 8 7 6 5 4 3 2 1 Arg Thr Pro Thr Glu Val Pro Arg Leu Met

Validation of assigned N-term methylation and glucuronidase sequence by Bottom-Up LC-MALDI-TOF/TOF analysis

Protein A L36Annotated ISD spectrum

The N-terminal sequence is b-gluronidase fused with protein A. The N-terminal Methionine is methylated. The N-terminal aminoacids not confirmed by ISD was confirmed by MS/MS of the N-terminal tryptic fragment

Results from MS Analyses

Please look at poster ##?? For more details

Lessons to be Learned from this Years StudyMass Spec Lessons..

1. Top-Down with ETD or ISD provides reliable N-term sequences

2. Top-Down CID was most easily misinterpreted

3. Edman and Top-Down Complement each other very well: Edman for the first ~10 residues, Top-Down for the inexpensive extension of calls (e.g. through the fusion site of Protein A)

4. Validation of the N-term by either T³-sequencing or Bottom-Up works as well

5. Efficient use of Top-Down MS requires good software support

6. Bottom-Up was great to confirm N-term results but not to generate them

7. Use of protein HPLC resulted in shortened readouts

8. Protein A Successful analysis of the fusion required high experience

9. Endostatin ragged N-termini were recognized by those that determined the intact molecular weight(s) , detected heterogeneity by HPLC or Edman

10.Top-Down by ETD or ISD permitted the detection of the C-terminal removal of Lysine, intact MW determination allowed to validate the finding

Next years ABRF-PSRG2013 studywhat's going to happen?

Most likely, the same proteins will be provided again! But: provided as a stew in a single pot! Task: Isolate/separate them from the mixture Problem: SDS-PAGE works well for Edman, but it is

difficult to extract intact proteins Hints:

Protein LC needs to be established, to get to the next level! Always try to get intact MW information! Use high sample amounts as you loose a lot during LC

The ABRF-PSRG Acknowledges the following Support

Recombinant Protein A was obtained as donation from RepliGen (Waltham, MA)

Endostatin was obtained as donation from SIGMA-ALDRICH (St Louis, MO)

Steve Smith (University of Texas Medical Branch) and Larry Dangott (Texas A&M University) for Edman sequencing to provide reference data for this study.

End

Following slides are bonus material

In-Source Decay (MALDI-ISD)

• “pseudo-MS/MS” technique, no precursor selection• ISD of protein in the MALDI plume at <nsec timescale (similar to ETD)• Fragmentation due to radical transfer from matrix to analyte (Takayama, 2001)• a,c- ions: N-terminus; y, z+2-ions: C-terminus – simultaneous sequencing• TOF/TOF allows for T³-sequencing: MS/MS analysis of ISD fragments

MALDI-ISD

MALDI-ISD and T³-Sequencing

Suckau & Resemann (2003) Anal Chem 75

ESI-ETD (Electron Transfer Dissociation)

CID• Collision with inert gas• protein is internally heated globally• it fragments in statistic process• weak bond cleavages

ETD• Collision with electron donating gas• perturbates electronic structure locally• resulting in local bond cleavages• ETD fragments all bond (except Pro)• for top down MS/MS of intact proteins

with precursor ion selection

ETD Measurement Cycle on QTOF

Reaction Cell

n-CI Source

10 kHz

1. Precursor Ion Accumulation2. Electron Transfer Reagent Addition3. ETD Reaction4. Fragment Ion Transfer and Detection

Tsybin et al. (2011) Anal Chem 83:8919

2245 2756 3267 3778 4289 4800

Mass (m/ z)

1505. 5

0

10

20

30

40

50

60

70

80

90

100

% Inten

sity

4700 Re flec tor Spec #1 MC[BP = 1364 .6 , 6319 ]

2320.021

2604.136

2476.098

2533.111

2994.245

2380.018

2719.152

2265.994

2511.039

2334.990

2866.211

3097.252

3244.298

2907.070

3501.516

3372.525

3334.382

3694.580

3571.589

3620.403

3850.483

3403.354

3777.469

3649.499

3727.686

4069.048

3954.714

3799.517

4104.137

4138.845

3897.704

4444.805

4195.564

4600.930

4296.463

4671.200

899. 0 1169. 2 1439. 4 1709. 6 1979. 8 2250. 0

Mass (m/ z)

6318. 9

0

10

20

30

40

50

60

70

80

90

100

% Inten

sity

4700 Re flec tor Spec #1 MC[BP = 1364 .6 , 6319 ]

1364.645

934.298

919.302

1862.823

1057.339

1661.772

1748.792

1250.611

1805.811

937.306

922.312

903.292

1993.845

1137.542

949.283

2149.937

967.460

1548.701

1121.476

1881.833

1893.830

1234.548

2206.957

1386.629

998.242

1014.250

1073.338

1089.329

1371.590

1060.379

1272.594

1034.432

1155.578

1767.799

981.249

1583.707

1102.528

1779.805

1202.542

1595.709

926.314

1171.544

1408.621

1313.632

1654.734

2178.956

1248.550

1471.685

911.340

1358.589

1666.741

1683.752

1828.800

1295.595

1484.663

1866.837

2190.964

1752.791

1505.684

2015.827

2065.896

1924.835

1427.620

2077.880

1452.631

1908.856

1792.781

1627.730

1936.783

I/L N I/L S G G M R G

N K/QD

FK/Q

C

E20ISD Endostatin (DAN): initial manual interpretation

Data base search for [IL]SGGMRGNR[KQ]DF[KQ]CF

Excerpt from COIA1_HUMAN

Excerpt from COIA1_MOUSE

Differences between human and mouse can be seen in the -2 position from the start of ISD sequence (ie. LNSPL in human and LNTPL in mouse)

Sequence from spectrum was found beginning at 1548.694, so we know there are a handful of residues preceding this seq

E20

9. 0 295. 4 581. 8 868. 2 1154. 6 1441. 0

Mass (m/ z)

360. 4

0

10

20

30

40

50

60

70

80

90

100

% Inten

sity

4 7 0 0 MS/MS Pre c urs or 1 3 6 4 .6 5 Spe c # 1 MC=>BC=>NF0 .7 [BP = 1 3 6 4 .6 , 3 6 0 ]

1364.60

110.09

70.10

101.09

23.03

86.12

1356.67

112.11

87.11

1371.48

665.30

72.11

235.10

391.13

197.12

1347.76

251.13

223.14

950.37

84.09

1049.43

169.13

44.09

837.30

115.11

1120.46

778.36

129.11

310.17

263.09

974.48

183.12

155.10

60.09

100.11

488.15

1234.51

30.07

280.11

534.24

1333.92

209.10

1317.66

587.19

1377.35

326.11

648.31

350.14

364.22

442.21

010212_B23_10pmol_Endostatin_MSMS_2kV_1364.65

To confirm N-terminus not covered in the ISD spectrum, MS/MS was performed on m/z1364.6

y7

b7 b8y9

b9 b10b2b3

b4 b5

Immonium Ions

P H

I/LK/Q

E20

y6

Determination of Endostatin N-termini by Edman degradation. - Major sequence matches CO1A1_HUMAN at position 1576. - A second sequence was found from position 1572. - Both sequences concur with the ISD findings.

E20

Edman sequencing detected the ragged N-term, ISD confirmed and extended itLargely manual analysis of ISD spectra made it difficult to extract full information

2012/2013 PSRG: Timeline of the 2-year study

ABRF 2011

ABRF 2012

Settled on the 3 standard proteins for distribution as separated proteinsi n year 1 of

the study

Year 1 (2012) Study

announcement

Samples sent to participants

Extended deadline

for returning data

Data analysis

Feb

‘11

Oct

‘11

Jan

‘12

Mar

‘12

May

‘12

Discussed ideas for 2012 study.Agreement upon a study design

May

‘11

Au

g ‘1

1

Sep

‘11

Feb

‘13

ABRF 2013

Distribution of proteins in mixture for year 2 of the study

Data analysisOct

‘12

Deadline for returning data

Jun

‘12

Year 2 (2013) Study

announcement

Comments……

un-reproducible recovery from the tube for Endostatin is a problem if one wants to optimize the setting or try to reproduce the data…..

Thanks! PSRG. It was fun. Unelss I've missed something, the availability of the proteins in the public domain made this an easy project. Sample quality was very good!

I thought the fusion Protein A solution was blocked? I obtained sequence matches to the protein B-Glucuronidase, either B-Glucuronidase is fused to Protein A and you were not successful blocking the protein or B-Glucuronidase is a contaminant……

It was very costly study for an Edman lab, (reagents).