1. Protein Sequencing Research Group: Results of the PSRG 2011 Study
Sensitivity assessment of Edman and Mass Spectrometric Terminal Sequencing of an undisclosed protein

2. Current PSRG Members Jim Walters (Chair) Sigma-Aldrich
J. Steve Smith University of Texas Medical Branch at Galveston
Wendy Sandoval Genentech, Inc.
Kwasi Mawuenyega Washington University School of Medicine
Bosong Xiang Monsanto, Co.
Detlev Suckau* Bruker Daltonics
Henriette Remmer* University of Michigan
Viswanatham Katta* Genentech, Inc
Peter Hunziker (ad hoc) University of Zurich
Jack Simpson (EB liaison) SAIC/National Cancer Institute at Frederick
* new members added in 2010

3. PSRG – Review
2009 Study – what techniques are complimentary to Edman – two samples
Edman remains reliable
MS based Top down techniques performed well with great promise and bottom-techniques successful when prior knowledge of sequence or reliable database information is available
2010 Study – follow on from 2009 using an antibody
It was necessary for ISD participants to use T3 sequencing to obtain true terminal information
Edman analyses required deblocking of the heavy chain
The most complete de novo sequencing was obtained by bottom up participants
Status: Edman sequencing and mass spectrometry based techniques have varied strengths and weaknesses depending on several experimental factors and both have a role in biochemical research
2010 Notables
Second year as PSRG and 3rd year for non-Edman participants
Three new members added
With a complimentary role realized, we attempt to push the capabilities of the varied sequencing techniques, namely assay sensitivity

4. 2011 PSRG Study Timeline Discussed ideas for 2011 study. ABRF
PSRG committee adds
three new members
explored different
potential study samples
ABRF
2011
Study Proposal
sent to EB
Settled on a designer
protein not in a database
2011 Study
announcement
Samples sent
to participants
Extended deadline
for returning data
Data
analysis
Mar ‘10
Sep
Jan ‘11
Feb ‘11
Oct ‘10
Discussed ideas for 2011 study.
Agreement upon a study design
Apr ‘10
May ‘10
Jun ‘10
Aug ‘10

5. PSRG 2011 Study Objective
To obtain terminal sequence information on varying amounts of a protein sample who’s sequence was not in a database

6. 2011 Study Design – The Sample Sets
Participants chose which of three sample sets they wanted to analyze (designated A, B or C)
Each sample set contained three tubes (designated 1,2 or 3).
Each tube contains the same recombinant protein with increasing amounts of material
Participants could request any single set (received 3 tubes), two sets (6 tubes), or all three sets (9 tubes)

7. The Protein Sample recombinant protein expressed in an E. coli system
molecular weight ~50 kDa
amino acid sequence of the protein is not in public domain database
sample was donated in liquid formulation in buffer
purified and AAA quantified

8. Sample Preparation and Distribution
A - lyophilized
Expressed protein purified using C-terminal His tag then by size exclusion chromatography and confirmed by SDS-PAGE.
protein containing fractions were quantified by AAA
dispensed into 1.5 mL tubes and lyophilized
dried samples were shipped as is, referred to as Set A.
or samples were resuspended and run on a gel (Set B) or pvdf membrane (Set C) and the gel/membrane slices corresponding to the ~50 kDa band were sent to participants.
the tube with lowest sample amount contains ~ 5 pmol dried, loaded on gel, or blotted on membrane
B – in gel samples
C – membrane 8

9. Requests of participants
Analyze samples in the designated numerical order or from lowest sample amount to highest and report on all samples analyzed
Edman sequencing: participants to provide amino acid yield data at every cycle
Alternative (MS based) methods: asked participants to provide the raw data files and peak lists, and method used for sequence assignment
instructed not to split sample due to the objective of the study and relatively low sample amounts
potential presence of a co-purified E. coli protein at <20 kDa in Sample Set A is known, but of no interest to current study.
suggested buffers to use to dissolve Sample Set A (lyophilized samples).
0.1 %TFA
25 mM ammonium bicarbonate
0.1% TFA / 20% acetonitrile
Participants asked to fill out a survey and all survey and raw data was submitted anonymously

10. 2011 PSRG Study Sample Set Requests

11. Survey response results (18 out of 38 Labs filled out a survey)

12. Survey response results

13. N-Terminal Techniques: Edman Degradation

14. Uses of Edman Sequencing
Cleavage site determination for proteases
Sequencing of MHC peptides
Sequencing of synthetic peptide libraries
Full characterization of proteins, especially recombinant proteins, that are present in large quantities
Stoichiometry, Edman is semi-quantitative
Protein identification for non-model organisms which do not have extensive DNA sequencing
Domain mapping
Confirmation of N-terminus
As a help for mass spectrometry sequencing to perform manual subtractions
Product characterization for SOPs for pharma
Can distinguish between the isobaric amino acids Leucine and Isoleucine
Clonality determination or antibody sequencing for cloning
Adapted from: ESRG Presentation: ABRF 2005

15. Solution (lyophilized)
Edman Workflows
PSRG 2011 Sample
Direct sequence
ABI Procise Instruments:
HT’s
cLC
Maximum # of correct calls from N-terminus reported
Sample Set
Sample Format
Sample Amount (pmols) 5 15 45 A
Solution (lyophilized) 24 32 49 B
Gel slice
N/A 9* C
Membrane piece 26 33
* no supporting data provided

16. Summary of Edman Data

17. Sample Sets A and C: N-terminal residues identified

18. Does increasing amount of sample increase calls?
Data trends toward longer reads as function of increased sample amount

19. Edman degradation sample solubility
Sample recovery was best when organic solvent was utilized.
Other solvents have been shown to be OK as well, data not shown.
Though most labs were able to determine the heavy chain was N-terminally blocked, only 3 labs chose to de-block the heavy chain and resequence
Reasons reported why labs did not deblock
No time
Enzyme too expensive

20. PSRG 2011 Edman Conclusions & Observations
Edman sequencing allows for direct determination of the protein’s N-terminal sequence.
Reliable N-terminal Edman data was obtained from the lowest concentration (5 pmol) samples for both Sample sets A and C.
Generally, slightly longer read lengths were noticed as sample concentration increased.
Sequencing preview and lag became more evident as sample concentration increased.
Contaminating proteins in the sample did not contribute negatively to any Edman result.
Sample A: concentration of contaminating protein was too low to be detected.
Sample C: sample was “isolated” by running the gel prior to blotting.
No C-terminal data was produced with Edman.
One lab returned N-terminal data from Set B (gel slice). Did not provide supporting data.
43 samples

21. N-Terminal Techniques Overview: Bottom-Up MS Techniques
Enzymatic Digestion

22. Uses of Bottom-up Sequencing
Protein identification via sequencing of unique (internal) peptides and subsequent database search
Biomarker discovery
A high degree of sequence coverage can be achieved by utilizing different proteases for digestion and combining results
Identification and localization of Post-translational Modifications
Identification and localizations of introduced protein modifications, e.g. cross linkers
Estimation of relative quantities of like proteins between samples via spectral counting
Confirmation of the complete protein sequence
De-novo elucidation of complete protein sequences
Elucidation of the N-and C-terminus with limitations (multiple enzymes or labeling strategies)
PSRG Presentation: ABRF 2011 22

23. Bottom-Up MS Experimental – LC-MS Systems
All Labs used LC separation prior to peptide analysis.
Eksigent NanoLC-2D
AB Sciex 4800
Thermo LTQ XL - 2
Thermo LTQ-Orbitrap Velos - 2
Bruker Ultraflex TOF/TOF

24. Bottom up Sample Preparation
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
m/z
909.34
518.27
631.36
274.30
389.23
840.14
525.30
794.34
437.01
507.89
939.12
548.38
725.28
205.06
891.38
596.10
175.25
872.45
578.15
822.11
679.19
320.14
728.67
707.15
482.91
402.96
661.30
256.01
440.89
386.13
316.13
967.97
742.47
455.25
215.14
PSRG 2011 Sample
MS and
MS/MS
100 mM AmBiC mM AmBiC
Digestion Enzymes
1 lab did Trypsin alone
Multiple enzymes
Trypsin, Glu-C, Lys-C
Trypsin, Glu-C
2 Trypsin, Chymotrypsin
Lys-C, Lys-N
2 MASCOT
3 manual
Data Explorer (AB)
Manual DeNovo Mascot
PEAKS 5.2
in house analysis software

25. Bottom up results 25

26. Bottom up Strategies – Lys-C/Lys-N digest:
A Novel Method for Analyzing Protein Terminals.
Kishimoto et. al., ASMS2010 TP08
Straightforward ladder sequencing of peptides using a lys-N metalloendopeptidase
Taouatas et. al., NATURE METHODS. VOL.5 NO.5., p ,2008
Lys-N Vendors: Associates of Cape Cod, East Falmouth, MA, Seikagaku KK, Japan
Proteome-wide analysis of protein carboxy termini: C terminomics. NATURE METHODS. VOL.7 NO.7. p , 2010
PSRG03 26

27. Comparison of N-terminal protein sequence of and Lys-C and Lys-N
Lys-N generates the same N-terminal peptide as Lys-C, except there is no lysine in the sequence for the Lys-N peptide.
Lys-N
PSRG03 27

28. Bottom up Strategies – Lys-C/Lys-N digest
Lys-N generates peptides with same m/z as Lys-C.
Exception 1.
no lysine in N-terminal
peptide using Lys-N
Exception 2.
No lysine in C-terminal
Peptide using Lys-C
Lys-N generates the same peptide as Lys-C, except there is no lysine in the sequence
PSRG03 28

29. C-terminal MS1 spectra from Lys-C digest
PSRG03 29

30. C-terminal peptide spectra and de novo sequencing
PSRG03 30

31. C-terminal peptide spectra and de novo sequencing
PSRG03 31

32. Combining Edman and enzymatic digestion using Trypsin and Glu C to identify N-term (Part #40)
Sequence Calls using Edman on Sample C3: GALRVFDEFKPLVEEPQNLIRVFDEFKPLVKPE
MS/MS Data using 4700
Participant 009

33. Bottom-Up Conclusions
Bottom up analysis involves enzymatic or chemical cleavage of the protein
followed by MS/MS analysis of the peptide mixture.
Small (6-25aa) fragments are generated that usually do not cover the complete protein sequence and may not include the terminal fragments.
Successful bottom up analyses utilized multiple enzymes and relied heavily on bioinformatics or manual data interpretation
Successful calling the N-terminus and C-terminus using lyophilized sample, 15 pmols
Successful calling C-terminus using in-gel sample, 15 pmol
MALDI and ESI show success as well as Orbitraps and TOF/TOF
Difficulty in assigning true N-terminal peptides however can used in complimentary fashion with Edman or dedicated chemistry to elucidate terminal peptides

34. N-Terminal Techniques Overview: Top-Down MS
In-Source Decay Fragmentation 34

35. In-Source Decay (MALDI-ISD)
MALDI-MS and MS/MS
MALDI-ISD
Analyte + matrix on metal target plate
Spot is excited with laser, ionization occurs
Ions are resolved by mass in TOF analyzer
Second TOF allows for MS/MS by precursor ion fragmentation
“pseudo-MS/MS” technique
Decomposition of protein in the MALDI plume at <nsec timescale
Ion formation due to radical transfer from matrix to analyte (Takayama, 2001)
Sequence determination without digestion (“Top Down”) even from large proteins
Second TOF allows for T³-sequencing
- Matrix generated hydrogen radical mediated fragmentation of the intact protein in the ion source via laser. 35

36. ISD and T3 Sequencing Suckau & Resemann, Anal Chem, Vol. 75, 21 (2003)36

37. Uses of MALDI-Top-Down Sequencing (ISD)
Confirmation of N-terminus, even if modified (pyroGlu, Methyl, Acetyl,…)
Confirmation of C terminus (terminal read length up to 80 residues)
Protein identification from low complexity mixtures
Biopharma: protein termini QC, side products elucidation (terminal truncations or elongations)
Fusion site confirmation in recombinant proteins
Proteolytic degradation product assignment
PTM elucidation; modification sites and types, PEGylation sites
Enzyme specificity testing on protein fragments (e.g. Kinase phosphorylation sites determination)
Full characterization of proteins that are present in large quantities
Full de novo sequencing capability up to ~15 kDa
Domain mapping
Identification of ragged termini
PSRG Presentation: ABRF 2011 37

38. ISD Experimental attempts
Matrix
Sample
Separation
ISD Instrumentation
0.1% TFA
20% ACN/0.1% TFA
DAN
1,5-diaminonapthalene
C4 ziptip
Bruker Ultraflex
Clean-Up
Chloroform-methanol precipitation
Recon in 0.1%TFA
DHB
2,5-dihydrobenzioc acid
AB Sciex 4800 38

39. Study Preparation: Cl-MeOH prec. ISDmanual data analysis
1975
2600
3225
3850
4475
5100
Mass (m/z)
217.7 5 10 30 35 40 45 50 55 60 65 70 75
% Intensity
4700 Reflector Spec #1 MC=>BC=>SM5[BP = , 721]
2087.4
2313.6
2200.5
2715.9
2412.7
2568.8
2057.4
2283.6
2524.8
2011.3
3109.1
2469.7
2862.1
2636.9
2168.5
2751.0
2408.7
3899.7
3742.5
3842.6
4125.9
4196.9
4253.0
4482.1
3983.8
4335.0
4593.8
4995.3
899.0
1117.2
1335.4
1553.6
1771.8
1990.0
721.4 80 90
100
1052.7
905.6
1619.1
1973.3
1041.6
927.5
995.6
1057.6
1091.7
978.6
907.6
1156.6
1277.9
1010.7
1845.3
1490.1
936.5
954.6
1562.1
1110.7
1037.7
1254.8
1863.2
1767.1
1901.3
MS/MS on 1619 F
[PK]
I/L V E
[PE]
K/Q
(G)
G A L R V F D E F K P L V E E
(N-terminal seq obtained from Edman analysis)
Red seq from ISD analysis b7 b8
b10 b4 b5
y10(?)
y11(?) N
I/L
I/L V R F R V

40. Summary of Top Down Analysis
None of the participants or PSRG succeeded in obtaining terminal sequences using ISD from study samples – other Top-Down methods were not attempted (ECD, ETD, …)
All participants did the routine things, but typical sample issues likely hindered analysis
Potential Reasons
Solubility - only a fraction of sample is recovered
Sample amount over estimated by traditional quantitative methods – less provided than presumed
Protein contamination has significant effect in Top-Down: problem and potential!
Limited sample availability: no investigation of problem, no optimization possible (intact MW, purity, solubility..)

41. Protein LC-separation of 100 pmol sample Pepswift PS-DVB (monolithic column)
100 pmol Casein
Result:
Several proteins present,
Much less protein available to the analysis than anticipated by original protein quantification
~ 5-10 pmol instead of 100 pmol
100 pmol study sample

42. Monolithic LC separation of Lyopholized sample
Protein of interest
Theoretical amount of 100 pmol
Reveals the presence of several proteins

43. ISD of Fraction 75 contains study sample: Matches sequence, but NOT de novo

44. ISD of Fraction 36 +Mascot: 30S ribosomal protein S15 E.coli

45. ISD of Fraction 32 +Mascot: YOBA_ECOLI Fragment 27-84

46. ISD of Fraction 47 +Mascot: HFQ_SERP5 N-term only (homolog to E. coli

47. Summary on MALDI-ISD study follow-up work
Expected ~50 kDa protein present plus contamination in the 16 kDa range
De novo sequencing was not possible due to sample amount restrictions
Protein LC-MALDI analysis showed only ~ 5-10 % of expected protein is available after separation
Multiple labs observed poor recovery from reverse phase columns
Protein LC-MALDI-ISD analysis theoretically starting with 100 pmols of sample
49 N-term and 56 C-term matches – not de novo – as sample amount was much lower than thought
IDs of several bacterial Heat Shock Proteins after ISD-Mascot analysis

48. Comments…’but not enough time’
I had planned to isolate/capture N-terminus but did not due to lack of time
Be more clear in instructions and allow much more time between sample arrival and data submission so that if extensive preparation is necessary, there will be time enough to perform it without affecting standard samples sequenced in the lab
Very nice setup; but I needed more time to take full advantage. As my ISD ambitions failed (!!) I turned to proteolytic digestions and PSD: Performed a lot of bottom up analyses, mainly after sulfonation…
Sorry, I did not have time to properly analyze the data and to do the experiment as if it would have to be done

49. Comments (continued)
did not spend time to purify or evaluate low level sequences by MS... Instructions were somewhat confusing. Not clear if the sample needed purification before Edman
Thanks! …even though we have de novo software we do NOT have a good strategy for obtaining sequence and determining N and C termini…Also, we identified quite a few peptides that likely weren't N-terminal or C-terminal…using other enzymes and finding overlapping sequences would have been a better strategy
I wouldn't mind trying another of these after I see how to approach it
I will be very interested in seeing the results of the mass spec analysis of these samples to which I do not have access…would like to see the comparison
It was very tough one to get the whole sequence even though it was not the goal
Sample has a ragged N-terminal sequence. ..Samples A1 to A3 were solublized in 01.% TFA and blotted but no sequence was observed…suggesting that no protein was in the tube or that it was insoluble in 0.1% TFA.
Challenging but good.

50. Final conclusions
Two techniques were successfully employed in this study to obtain N-terminal sequence of an undisclosed protein not present in public databases.
Edman Degradation – lowest sample amounts of Samples A and C
Enzymatic Digestions – 15 pmols sample amounts of Sample A and B
For Edman, slightly longer read lengths were noticed as sample concentration increased, however, sequencing preview and lag became more evident.
De novo Bottom-up was not successful unless a priori knowledge of sequence was obtained (by Edman, database…etc). There are strategies which can be successful however the current strategies have limitations.
For Top Down, not successful in obtaining terminal sequences using ISD from study samples – other Top-Down methods were not attempted.
Likely reasons: poor recovery due to solubility, hindering impurities, Ionization, etc.
Top down was able to obtain sequence in 100 pmol sample using protein LC and MALDI-ISD strategy as long as theoretical sequence was utilized.
Time is of the essence – for committee to appropriately design and develop study and for participants to be able to properly analyze samples.
De-novo elucidation of the complete protein sequence is limited since N-and C-termini cannot be identified in straightforward fashion
Elucidation of the N-and C-terminus requires a dedicated experimental strategy, specialized chemistry (e.g. combination of LysC and LysN), extensive manual data interpretation and an experienced scientist
A combination of bottom-up and terminal sequencing (Edman or top-down) allows for comprehensive de-novo protein characterization

51. Acknowledgements Participating labs!!!!!!!
Robert English University of Texas, Medical Branch at Galveston
Accumulation & annonimization of data
Shantanu Roychowdhury - Sigma-Aldrich
Expressed and purified protein
Anja Resemann - Bruker Daltonics
LC MALDI ISD and Top Down work
Jack Simpson and the rest of ABRF Executive Board
For support and scrutiny of study proposal
Participating labs!!!!!!!