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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).