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Terminal Sequencing of Standard Proteins in a Mixture. Protein Sequencing Research Group (PSRG): Results of the PSRG 2012/13 Study Year 2. PSRG Members. Current Members Greg Cavey Southwest Michigan Innovation Center Robert English (Co-Chair) University of Texas Medical Branch

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psrg members
PSRG Members

Current Members

  • Greg CaveySouthwest Michigan Innovation Center
  • Robert English (Co-Chair) University of Texas Medical Branch
  • Mark GarfieldNIH/NIAID
  • PegahJaliliSigma-Aldrich
  • Sara McGrath (Co-Chair)FDA
  • EjvindMortzAlphalyse

Outgoing Members

  • Henriette Remmer (Co-Chair)University of Michigan
  • Jack Simpson (EB liaison)United States Pharmacopeia
  • Detlev SuckauBrukerDaltonics
  • Jim Walters (ad-hoc)Sigma-Aldrich
  • ViswanathamKattaGenentech, Inc

Staying on as ad-hoc member for the coming year: Henriette Remmer

study background and design
Study Background and Design

Status of Terminal Sequencing

  • N-terminal sequencing is in the midst of a technology transition from classical Edman sequencing to mass spectrometry (MS)-based sequencing.
  • Edman sequencing and MS-based techniques both have strengths and weaknesses.
  • With a complimentary role realized, the PSRG attempts to push the capabilities of the various sequencing techniques, namely terminal sequencing of proteins in a mixture.

Concept of the Study - Terminal Sequencing of Proteins in a Mixture

  • Sequencing proteins in a mixture typically requires separation of proteins prior to analysis for both Edman sequencing and MS-based technology platforms.
  • Edman Sequencing : SDS-PAGE and electroblotting of the separated proteins – well established everywhere
  • MS-based sequencing:LC separation necessary prior to analysis
  • – not well established in most core facilities

PSRG designed a 2-year study

  • YEAR 1: Terminal sequencing and I.D. of three separatedstandard proteins
  • YEAR 2: Proteins (+ one new protein) distributedin a Mixture
last year s study objective
Last Year’s Study Objective

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

last year s study design the samples
Last Year’s Study Design – The Samples
  • Participants were asked to analyze samples for terminal sequencing using any technology available.
  • Participants received all three proteins with ID in sufficient amounts to sequence each protein utilizing all three technologies. Feasibility of analysis had been previously validated by PSRG members.
  • Participants also filled out a survey, all responses were kept anonymous.
last year s participation survey results
Last Year’s Participation & 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 1 bottom-up MS (5 used bottom-up for confirmation).
  • Out of 14 respondents,
    • 9 labs analyzed the reference protein BSA, 8 correctly determined the N-terminus.
    • 13 labs analyzed Protein A , 4 correctly determined the N-terminus (methyl-Met).
    • 14 labs analyzed Endostatin, 12 labs correctly determined the N-terminus , only 7 identified the presence of the second N-terminus.
last year s edman summary observations
Last Year’s Edman Summary &Observations

Edman sequencing allows for direct determination of

the N-terminal sequence of a protein.

  • All labs returned N-terminal data which correlated well with published protein sequences.
  • Edman can produce data with and without separation (SDS PAGE and chromatography).
  • No C-terminal data is produced with Edman.
  • If the protein is N-terminally blocked, the reaction will not proceed for most (but not all) modifications.
ms lessons learned from last year
MS Lessons Learned from Last Year
  • Top-Downwith ETD or ISD provided reliable N-term sequences
  • Top-Down CID was most easily misinterpreted
  • 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)
  • Validation of the N-term by either T³-sequencing or Bottom-Up
  • Efficient use of Top-Down MS requires good software support
  • Bottom-Up was great to confirm N-term results, but not to generate them
  • Use of protein HPLCresulted in shortened readouts
  • Protein A: Successful analysis of the fusion protein required high experience
  • Endostatin ragged N-termini were recognized by those that determined the intact molecular weight(s) , detected heterogeneity by HPLC or Edman
  • Top-Down by ETD or ISD permitted the detection of the C-terminal removal of Lysine, intact MW determination allowed validation of the finding
slide9

PSRG 2012/13 (Year 2/2) Study Objective

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

in amixture

psrg 2012 13 study design the samples
PSRG 2012/13 Study Design – The Samples
  • Each participant received 2 vials of the mixture (except one late participant), and each vial contained the protein amounts listed above with buffer components including 4M urea and PBS. Each participant also received a third vial containing 750 pmol BSA.
  • Participants were asked to a) separate the proteins, and b) analyze samples for terminal sequencing using any technology available.
  • All protein components showed solubility in traditional proteomic buffers, including water, 0.1% FA, and 0.1% TFA. Specifically, endostatinhad shown solubility in 20% ACN, 0.1% TFA, 50% pyridine, and buffers compatible with 1D gel electrophoresis.
  • Participants also filled out an online survey (responses were kept anonymous).
  • * Protein not included in last year’s study.
protein n termini
Protein N-termini
  • Two participants, 16X and 20M submitted Edman sequencing results.
  • Participant 16X tried different techniques for protein separation
  • The PSRG used Edman sequencing without prior separation of proteins for comparison
edman sample preparation workflows
Edman Sample Preparation Workflows

PSRG 2013 Samples

Gel Eluted Liquid Fractionation Entrapment Electrophoresis (GELFrEE)

(16X)

Used sample as

Provided

-no separation-

(PSRG)

SDS PAGE –

blotting on PVDF

(16X, 20M)

HPLC

(16X)

ABI Procise

2- 494 HT

1 – 492 HT

slide13

16 X

Edman Results

Entire sample tube A used for HPLC:

50% of fractions 7, 10 and 11 for Edman sequencing:

30 pmol Protein A

150pmol Endostatin

400 pmola-S1 Casein

Fraction 10

Fraction 11

Fraction 7

Fraction 10: no amino acid assignments

slide14

Edman Results

16 X

90% of sample tube B separated on GELFrEE tube gel eluter :

GELFrEE System: Gel Eluted Liquid Fractionation Entrapment Electrophoresis

  • Disposable cartridges contain SDS-polyacrylamide gel matrix
  • Proteins are solubilized and electrophoresed
  • Size based separation and liquid phase recovery
  • Sample Preparation after GELFrEE separation:
  • Fractions evaporated to dryness
  • Konigsberg acetone precipitation1 (3x) followed by 2 acetone washes
  • Dissolved precipitate in 0.1% TFA
  • 50% of solution for Edman sequencing-applied to Glass Fiber Filter with polybrene treatment
  • 27 pmol Protein A; 135 pmolEndostatin , 360 pmola-S1Casein

No meaningful Edman data obtained –only Gly and Tris artifact peaks

1 LE Henderson, S Oroszlan, W Konigsberg; Anal. Biochem. 1979, 93(1), 153-157

slide15

Edman Results

20M

30% of one sample tube used with sample preparation by SDS-PAGE/pvdf blotting:

20 pmol Protein A; 100 pmolEndostatin , 250 pmola-S1Casein

12 of 15 residues correctly assigned

15 residues correctly assigned

25 residues correctly assigned

slide16

Edman Results

PSRG

Sequencing of protein mix without prior separation

Started with 30% of the mix from one tube: 15 pmol Protein A, 80 pmolEndostatin, 200 pmola-S1 casein

Repetitive Yield: 95%

slide18

Effectiveness of HPLC for sample preparation

compared to SDS-PAGE/blotting

psrg 2013 edman conclusions observations
No one detected all 4 N-termini by Edman sequencing of the separated proteins

SDS/PAGE/electroblotting to pvdfwas the most successful for sample preparation

HPLC can have preparative losses and electroelutionbuffer interference

Edman sequencing can produce data without prior separation of proteins, but for complex mixtures, protein separation is necessary

If the protein is N-terminally modified, the reaction will not proceed for most modifications

PSRG 2013 Edman Conclusions & Observations
what is top down mass spectrometry why is it most appropriate for terminal protein sequencing
What is Top-Down Mass Spectrometry?why is it Most Appropriate for Terminal Protein Sequencing?
  • All MS Analysis based on intact undigested protein
  • Intact molecular weight: gross structure validation
  • Targeted sequencing of the N- and C-terminus
  • Bottom-up analysis tackles the termini only arbitrarily

N-TERMSEQUENCE

C-TERMSEQUENCE

most employed terminal sequencing in psrg studies are base on maldi in source decay maldi isd

LASER

N

M

N

T

R

M

MALDI

Intact Sequencing

N

T

E

E

R

M

N

T

E

R

T

E

R

M

N

T

E

R

M

C

T

E

R

M

N

T

E

R

M

S

E

C

T

E

R

M

N

T

E

R

M

S

E

C

E

C

T

E

R

M

Most employed Terminal Sequencing in PSRG Studies are base on MALDI-In Source Decay (MALDI-ISD)

MALDI N & C-Terminal Sequencing

1,5-DAN

N

T

E

R

M

S

E

Q

U

E

N

C

E

C

T

E

R

M

MALDI TOF/TOF

40,000 resolution

30 seconds!

  • Confirm N & C terminal sequences
  • Identify truncations/terminal PTMs
  • Generate sequence information without proteolytic digestion
  • In less than 1 minute
slide23

K

Intact Panitumumab N & C-Terminal Sequencing – 1 ISD Spectrum > 2 Protein Sequencessequence match of LC confirming the sequence of both termini

64

21

slide24

pyroGlu-

X

Lys-truncation

K

Intact Panitumumab N & C-Terminal Sequencing 1 ISD Spectrum > 2 Protein Sequences sequence match of HC confirming N-term pyro-glutamylation and C-term lysine truncation

72

50

panitumumab sequence covered in a single maldi isd spectrum

Heavy Chain

Q/pE (-18 Da)

Q/pE (-18 Da)

FR1

FR1

CDR1

CDR1

FR2

FR2

CDR2

FR1

CDR2

FR3

FR3

FR1

CDR3

CDR1

CDR3

FR2

CDR1

FR4

FR4

FR2

CDR2

CDR2

FR3

FR3

CDR3

CDR3

FR4

FR4

CH1

CH1

CL

CL

Hi

Hi

Light Chain

C

C

18 C-C

(- 36Da)

H

H

N295ST

2

2

glycosylation

N

-

C

C

H

H

3

3

0,1, 2 K

Panitumumab Sequence Covered in a Single MALDI-ISD Spectrum

c-ions

z,y-ions

sequence covered after lc separation maldi tds middle down increases sequence coverage for mabs

Heavy Chain

Q/pE (-18 Da)

Q/pE (-18 Da)

FR1

FR1

CDR1

CDR1

FR2

FR2

CDR2

FR1

CDR2

FR3

FR3

FR1

CDR3

CDR1

CDR3

FR2

CDR1

FR4

FR4

FR2

CDR2

CDR2

FR3

FR3

CDR3

CDR3

FR4

FR4

CH1

CH1

CL

CL

Hi

Hi

Light Chain

C

C

18 C-C

(- 36Da)

H

H

N295ST

2

2

glycosylation

N

-

C

C

H

H

3

3

0,1, 2 K

Sequence Covered after LC-SeparationMALDI-TDS Middle-Down Increases Sequence Coverage for mAbs

c-ions

z,y-ions

fast and extensive mab product characterization middle down panitumumab analysis

HC

Fd

LC

Fc/2

3.Full TCEP reduction

1. Endoglycosidase F2

2.IdeS cleavage

Fast and Extensive mAb Product Characterization Middle-Down Panitumumab Analysis

1. Remove glycan, 2. cleave HC, 3. reduce

  • IdeScleaves the HC of mAbs specifically at a conserved Gly-Gly motif in the hinge region
  • Works for most mammalian antibodies in their native form
slide28

Middle-Down Analysis of IdeS Digest

Panitumumab: LC-MALDI

Fd

(M+H)1+

(M+2H)2+

LC-MALDI-TDS Analysis of Panitumumab

Fabricator Digest

Column: Zorbax C8 Matrix: sDHB

R(t)

(M+3H)3+

(M+2H)2+

(M+H)1+

LC

Fc

slide29

Middle-Down Analysis of IdeS Digest

PanitumumabIdeS+GlycoZERO: TDS of Fc-Fragment

LC-MALDI-TDS

Fc Fragment

Localization of glycan

C-terminal K truncation

Coverage: 62%

slide30

Middle-Down Analysis of IdeS Digest

Panitumumab: TDS of LC

LC-MALDI-TDS Light Chain:

90 AA from N-terminusCoverage: 70 %

slide31

Middle-Down Analysis of IdeS Digest

Panitumumab: TDS of Fd-Fragment

LC-MALDI-TDSFd Fragment

91 AA of variable N-terminus

Coverage: 58 %

considerations for top down lc maldi isd analysis
Considerations for Top-Down LC-MALDI-ISD Analysis
  • ≥ 50 pmol protein/LC-fraction is desired
  • ≥ 100 pmol/protein need to be applied to column for each protein
  • i.e., a 10 protein mix loads > 1 nmol on column
  • Suitable columns: C8capLC, monolithic columns
  • Severe protein losses typical during LC decrease signal
  • Reduction/alkylation and oxid. typically increase noise
software for top down sequence analysis
Software for Top-Down Sequence Analysis
  • Functionality
  • Assign expected sequence to TDS spectrum (BioPharma QC)
  • ID Proteins through TD standard Mascot searches (Discovery, ID)
  • Manual/Automatic de novo sequencing (Edman like)
  • Manual/Automatic de novo sequencing + BLAST (Sequencing+ID)
  • Generate test hypothesis to explain Dm´s (terminal mods, truncations)
  • Available software
  • BioTools 3.2 (Bruker Daltonik ECD/ETD/MALDI-ISD)
  • ProSight PTM (Kelleher Group ETD)
  • ProSightPC 2.0 (Thermo ETD)
slide34

MALDI Top-Down andMiddle-Down Analysis in routineBioPharmaQC

  • Intact Protein MW < 10 ppm for MW upto 30 kDa
  • Middle Down antibodyworkideallysuitedfor MALDI
    • Screen forPTMsorprocessingerrorson thedomainlevel
    • Validateprocessingerrorsby MALDI-TDS
    • Automatedscreening /validation in BioPharmaCompass
    • MWs in minutes
    • TDS in sec underfullautomation
maldi isd fragments and database searching y z 2 15 01 da c a 45 02 da
MALDI-ISD Fragments and Database Searchingy-(z+2) = 15.01 Da, c-a= 45.02 Da
  • Top-Down Search principle
  • Select m/z range of ISD fragments as “virtual precursor ions”
  • lower mass fragments are used as dependent fragments
  • Standard MS/MS Mascot search with MALDI-ISD as fragment ion set
maldi tds result from b galactosidase terminal sequences confirmed in seconds
MALDI-TDS Resultfromb-GalactosidaseTerminal SequencesConfirmed in Seconds..

N-termMethioninetruncationconfirmed

manual de novo sequencing
Manual de novo Sequencing
  • c-ion series assigned by:
  • High intensity
  • -45 Da a-ion satellites
  • Proline gaps
maldi tds de novo analysis of the n terminus
MALDI-TDS De Novo Analysis of the N-terminus

Resemann et al. 2010 Anal Chem 82:3283-92

Top-down de novo protein sequencing of a 13.6 kDa camelid single heavy chain antibody by MALDI-TOF/TOF MS.

Resemann et al. 2010 AC

cap lc monolithic column 10 sample loaded dionex ps dvb 500
Cap-LC (monolithiccolumn, 10 % sample loaded)Dionex PS-DVB 500

Protein A intact

UV LC traces allow to quantify

Proteins in unknown samples

His-Tag Casein

Protein A

truncation forms

Endostatins

slide41

LC-MALDI MS Analysis of the PSRG2013 Sample

Endostatins

His-Tag Casein

Protein A intact

Protein A truncation forms

workflow needed this year for ms analysis
Workflow needed this year for MS analysis
  • Separate proteins by LC
  • Determine Protein MW after LC separation
  • Assign Protein IDs based on MW (sequences known)
  • Establish Top-Down sequence analysis online or offline
  • Map experimental terminal sequences to the known protein sequences
ms strategies used in 2013 study
MS Strategies Used in 2013 Study

Most respondents

Additional work by some

Trypsin digest of fractions containing proteins

CID or ISD for sequence determination (bottom-up)

Some needed more sample cleanup or SDS-PAGE visualization of protein fractions

  • HPLC of intact proteins with fraction collection
  • LC-UV or ESI-MS to determine fractions of interest
  • Intact MW by MALDI or ESI
  • Sequence determination by ISD (top-down)
goal 1 good separation detect expected proteins
Goal 1 = good separation, detect expected proteins
  • Data provided shows good protein separation
  • First use intact MW to detect known proteins
    • MALDI MW usually very accurate
    • ESI MW determination possible with deconvolution software
    • Poor S/N or modifications complicate interpretation
  • Most intact MW data provided is good enough to indicate protein variants
    • Cannot ID specific differences
    • Probably not sufficient for samples with unknown proteins
successful protein separation
Successful Protein Separation

08D

  • HPLC system: Agilent 1200
  • Column: Agilent Poroshell 300SB-C8, 2.1x75 mm 5-micron
  • Solvent A: 0.1% TFA in water, Solvent B: 0.1% TFA in ACN
  • Gradient: 25-60% B in 9.5 min, Flow: 0.5 ml/min, Column temperature: 70°C

12P

  • HPLC system: Thermo Surveyor
  • Column: Waters Biosuite Phenyl 1000 2.0x75 mm 10-micron
  • Solvent A: 0.1% FA in water, Solvent B: 0.1% FA in ACN
  • Gradient: 5-95% B in 60 min, Flow: 0.1 ml/min

16X

  • HPLC system: Agilent 1260-Dionex Chromeleon
  • Column: Zorbax 300SB-C8 2.1x150mm
  • Solvent A: 0.1% TFA in water, Solvent B: 0.1% TFA in ACN
  • Gradient: 5-95% B in 40 min, Flow: 0.2 ml/min, Column temperature: 40°C

16X

  • HPLC system: Agilent ChipCube
  • Acquisition conditions unknown
    • Agilent does offer Intact Protein Chip
    • C-8 SB-ZORBAX, 300Å, 75 μm x 43 mm
identification by intact mw endostatin

08D

Identification by Intact MW – Endostatin

MALDI of fractions 6-9 shows protein corresponding to Endostatin in all

Variant 2 only appears in fraction 6

5

4

8

6

9

1

7

2

3

  • HPLC system: Agilent 1200
  • Column: Agilent Poroshell 300SB-C8 2.1x75 mm 5-micron
  • Solvent A: 0.1% TFA in water
  • Solvent B: 0.1% TFA in ACN
  • Gradient: 25-60% B in 9.5 min
  • Flow: 0.5 ml/min
  • Column temperature: 70°C
  • MS: BrukerAutoflex Speed
  • MALDI /DHB matrix for intact MW
identification by intact mw endostatin1

16X

Identification by Intact MW – Endostatin

ESI-MS chromatogram highlighting 3rd peak

Mulitply-charged ESI envelope with good S/N – easy to interpret

Instrument software allows deisotoping, intact MW, and determination of variants

  • HPLC system: Agilent ChipCube
  • MS: Agilent 6210 ESI-TOF
  • Acquisition conditions unknown
identification by intact mw protein a

12P

Identification by Intact MW – Protein A

ESI-MS chromatogram highlighting 1st peak

Mulitply-charged ESI envelope with poor S/N –harder to see by eye

Instrument software deisotoping and intact MW determination still good for this protein

  • HPLC system: Thermo Surveyor
  • Column: Waters Biosuite Phenyl 1000 2.0x75mm 10um
  • Solvent A: 0.1% FA in water, Solvent B: 0.1% FA in ACN
  • Gradient: 5-95% B in 60 min, Flow: 0.1 ml/min
  • MS: LTQ-FT Ultra
  • ESI infusion for intact MW
accurate n term sequence protein a

16X

Accurate N-term sequence – Protein A

G

D

  • Sample: 0.1% TFA + 50 mg/mL TCEP
  • Mix 1:1 with 10 mg/mL DAN matrix
  • MS: BrukerUltraflex TOF/TOF ("A")
  • MALDI/ISD top-down
accurate n term sequence protein a1

08D

Accurate N-term sequence – Protein A

Methylation of N-terminal methionine

  • MS: BrukerAutoflex Speed
  • MALDI /DHB matrix
  • Fractions for ISD selected based on intact MW results
sample manipulation
Sample Manipulation
  • Sample simplified by reduction/alkylation
    • aS1 Casein observed as a dimer (3 Cys protein)
    • Reduction of the sample resulted in simplified chromatogram
    • Some respondents use reduction as a matter of protocol
  • However, less sample manipulation is better
    • Sample loss at every step
    • Oxidation or other artificial modifications to the proteins can occur
identification by intact mw a s1 casein

12P

Identification by Intact MW – aS1 Casein

ESI-MS chromatogram highlighting 3rd peak

Mulitply-charged ESI envelope and deconvolution to intact MW

Protein appears as a dimer – greater chance of misidentification?

  • HPLC system: Thermo Surveyor
  • Column: Waters Biosuite Phenyl 1000 2.0x75mm 10um
  • Solvent A: 0.1% FA in water, Solvent B: 0.1% FA in ACN
  • Gradient: 5-95% B in 60 min, Flow: 0.1 ml/min
  • MS: LTQ-FT Ultra with ESI infusion
  • Deconvolution for intact MW by ThermoFisher ProMass software
chromatogram simplified by reduction

08D

Chromatogram Simplified by Reduction

Native

Reduced and alkylated

5

2

4

8

1

6

9

1

7

2

3

4

3

5

The native fraction 5 peak (RT = 6.7 min) disappears after reduction and the RT = 5.3 min peak increases. The reduction cleaves the dimer.

  • HPLC system: Agilent 1200, Column: Agilent Poroshell 300SB-C8 , 2.1x75 mm 5-micron
  • Solvent A: 0.1% TFA in water, Solvent B: 0.1% TFA in ACN
  • Gradient: 25-60% B in 9.5 min, Flow: 0.5 ml/min, Column temperature: 70°C
  • UV detection
cid or isd identification a s1 casein

08D

CID or ISD Identification – aS1 Casein

MALDI-ISD (reduced), intact protein

Correct N-term identification possible on native protein by top-down MS

Bottom-up MS provides confirmation that N-term identification is correct

ESI-CID (native), tryptic peptide

goal 2 accurate n terminal sequence

Endostatin 1 C-ox 19445.871 Da

Endostatin 2 C-ox 19963.414 Da

Protein A 44612.0 Da    

aS1-casein dimer 50011.2 Da     

Goal 2 = Accurate N-terminal sequence
mass spec techniques problems comments
Mass Spec - Techniques, Problems, Comments
  • Proteins collected in fractions may precipitate
    • Several respondents mentioned poor results for ESI/CID analysis of samples that had been HPLC-separated and fraction-collected
    • Led to fewer confident N-terminal identifications
    • Problem not observed by participants using MALDI-ISD analysis (more experience with the workflow?)
mass spec techniques problems comments1
Mass Spec - Techniques, Problems, Comments
  • Not enough material?
    • “There was not much sample for method development. There was no room for mistakes, which was very challenging.”
  • In 2012 study, 100 pmol provided by PSRG, 30-100 pmol used by respondents

(quantity provided by PSRG)

60

300

800

mass spec techniques problems comments2
Mass Spec - Techniques, Problems, Comments
  • Not enough material?
    • “There was not much sample for method development. There was no room for mistakes, which was very challenging.”
  • In 2012 study, 30-100 pmol used by respondents
  • However, in 2013 study, all participants needed protein separation
    • Significant loss due to separation techniques, multiple injections needed?

5

3

What methods, if any, did you use to separate and purify the sample?

2

10

1

2012

2013

mass spec techniques problems comments3
Mass Spec - Techniques, Problems, Comments
  • Analysis
    • All participants provided intact MW information
      • One of the lessons from last year 
    • Only repeat participants provided correct sequence information
      • Top-down MALDI-ISD appears to be best to sequence N-terminus
      • Bottom-up if needed to confirm N-or C-terminus
  • Skill level may be a factor in successful analysis
    • No information on the skill level of participants
    • How many participants determine N-terminal sequences on a regular basis?
    • We did not get comments on how these study samples compare to regular client samples
    • Intact protein separation in MS-based workflows is still uncommon in most core labs, but top-down proteomics is on the rise
study conclusions
Study Conclusions
  • Edman sequencing allows direct determination of the N-terminus
    • Prior protein separation is necessary for a protein mixture.
  • Top-down MS with MALDI-ISD provided reliable N-terminal sequences
    • Further validated by bottom-up MS
    • Prior protein separation by HPLC works reasonably well
  • Edmanand Top-Down complement each other very well
    • Edmanfor the first ~10 residues
    • Top-Down for extension of calls (e.g. through the fusion site of Protein A).
  • Use ofHPLC separation posed a significant challenge to all labs
    • For Edmanas well as top down MS
    • This workflow needs work to be established in core labs
    • Be aware of losses and chemistry artifacts due to oxidation or reduction/alkylation
  • Minor protein variants in a mixture are overlooked by both techniques
    • Report intact MW
    • Attempt Edmansequencing with prior protein separation.
acknowledgments
Acknowledgments

Sponsors of Study Proteins:

  • ABRF
  • Biomedical Research Core Facilities, University of Michigan
  • Repligen
  • BrukerDaltonics
  • Sigma Aldrich

Anonymizer:

  • XuemeiLuo, University of Texas Medical Branch

Edman Sequencing: Steve Smith, University of Texas Medical Branch

MALDI-ISD: Anja Resemann, Bruker Daltonics

………and study participants!!!!!!

proposal for 2014 study
Proposal for 2014 Study
  • Focus on protein separation techniques for terminal sequencing of a simple protein mixture
  • ABRF-wide Edman sequencing survey
  • Other ideas?????
slide67

If YESto question #4, how much material did you use in order to confidently call the N-terminus of the protein ? Please specify units.

slide68

If NOto #4, how much more material would you need in order to confidently call the N-terminus of the protein? Please specify units.

slide69

Please list the amino acid sequence (in single letter code) that you were able to CONFIDENTLY call for the N-terminus of the protein

successful protein separation1
Successful Protein Separation

HPLC-ESI

HPLC-UV

5

4

8

6

9

1

7

2

3

HPLC-DAD