Protein Sequencing Research Group ABRF 2015 annual meeting

» N-terminal sequencing is in the midst of a technology transition from classical Edman sequencing to mass spectrometry (MS)-based sequencing

» Core laboratories need to have well-defined protocols for terminal sequence analysis by MS

» Practice with types of samples, sample preparation protocols and expected results are critical

» N-terminal sequencing is in the midst of a technology transition from classical Edman sequencing to mass spectrometry (MS)-based sequencing

» Core laboratories need to have well-defined protocols for terminal sequence analysis by MS

» Practice with types of samples, sample preparation protocols and expected results are critical

» This year’s study: use dimethylation of protein to

enhance identification of N-terminal peptide sequence ˃ 1 known purified protein (myoglobin) ˃ Successfully derivitize the proteins using a provided protocol ˃ Digest and identify the derivitized peptide fragments by MS ˃ Determine amount of protein needed for practical use

» 25 laboratories requested samples ˃ 10/25 (40%) international sites

» 13 participants returned data, 4 reported incomplete results

» All participants reported this as a challenging study

7

1 2 3

No Yes, a little Yes, a lot n/a

Do you have experience with N-term labeling?

18%

82%

Did you particpate in the 2014 study?

YesNo

» Identify the N-terminus of known proteins using chemical derivitization (dimethylation)

» Determine lowest amount of protein identified

Workflow (A) In-solution labeling

Workflow (B) In-gel labeling

Chemical labeling of proteins at N-terminus SDS-PAGE separation of proteins*

Cleanup with SDS-PAGE or membrane cut-off filter

In-gel chemical labeling

Digestion in-gel or on cut-off filter In-gel tryptic digestion and cleanup

MS analysis including data analysis MS analysis including data analysis

Identification of N-termini Identification of N-termini

*This study used only a single purified protein

7

1

2

2

1

1

0 1 2 3 4 5 6 7 8

Orbitrap

Bruker Ultraflex

AB Sciex 4800/5800 TOF

AB Triple Tof

Axima QIT-TOF

Synapt

What instrumentation did you use?

Workflow A

54%

Workflow B

31%

Both A and B 15%

Which Workflow did you perform?

Reaction conditions Dimethyl labeling » 5uL of 1pmol/uL protein (5pmol) » 100mM Na acetate, pH 5 » 4% formaldehyde » Na cyanoborohydride » Incubate at RT

» Addition of dimethyl by reductive amidation

» Reacts with N-term and Lysine residues

NH2-peptide N-peptide NaBH3CN

CH3

CH3

║ H H

O

GLSDGEWQQVLNVWGKVEADIAGHGQEVLIRLFTGHPETLEKFDKFKHLKTEAEMKASEDLKKHGTVVLTALGGILKKKGHHEAELKPLAQSHATKHKIPIKYLEFISDAIIHVLHSKHPGDFGADAQGAMTKALELFRNDIAAKYKELGFQG

Myoglobin: P68082

» Nt-dimethyl peptides are best sequenced from b ions

H3C

OHN C C N C C N C C N C C

R1 R2 R3 R4

OO O OH3C H H H H H H H

b1 b2 b3

y3 y2 y1

y ions do not contain N-terminal information

» Nt-dimethyl peptides are best sequenced from b ions » Internal dimethylations can confound results

H3C


R1 R2 R3 R4


b1 b2 b3

K H3C CH3

» Nt-dimethyl peptides are best sequenced from b ions » Internal dimethylations can confound results

H3C


R1 R2 R3 R4


b1 b2 b3

b3 ion is not conclusive – dimethyl can be at either N-terminus or R3

Must verify that b ions indicate correct placement of dimethyl group

K H3C CH3

» SDS-PAGE purified » In-gel derivatized » In-gel digested

Myoglobin ~17 kDa

5 pmol 85 ng

3 pmol 51 ng

1 pmol 17 ng

0.3 pmol 5 ng

5 0.3 0.3 3 3 3 3 0.3 0.3 5 Myoglobin

SDS-PAGE

Amount Loaded (pmol)

Underivatized Peptide

Dimethyl Nt Peptide

Dimethyl Nt, 1xDeam Peptide

Dimethyl Nt, 2xDeam Peptide

Base Peak

Extracted Ions for GLSDGEWQQVLNVWGK:

Extracted Ions for GLSDGEWQQVLNVWGK:

Estimation of Dimethylated Myoglobin

GLSDGEWQQVLNVWGK m/z 908.4539 (2+)

DiMethyl-GLSDGEWQQVLNVWGK m/z 922.4694 (2+)

» Most participants were successful with 5 pmol starting material

» Several could detect 0.3 pmol of starting protein

1

2

3

2

1 1 1

2

0 0

Workflow A (in-solution labeling) Workflow B (in-gel labeling)

Lowest Quantity of Protein Detected

more 5 pmol 1 pmol 0.3 pmol less

In this sample, only the N-terminal peptide was labeled on N-terminus

Spectrum of Labeled N-terminal Peptide Most b ions are represented

920 921 922 923 924 925 926 927 928m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

922.97z=2922.47

z=2

923.47z=2

923.97z=2

927.98z=?

928.3z=?

921.16z=?

924.81z=?

927.48z=?

926.66z=?

920.41z=?

924.30z=?

922.31z=?

925.85z=?

920 921 922 923 924 925 926 927 928m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

922.47

922.97

923.47

927.92

927.42926.27920.89 923.97 924.85 9922.41 926.89

Dimethyl-GLSDGEWQQVLNVWGK [M+2H]2+ at m/z 922.47

5 pmol 1 pmol

» Detection of peptide at MS1 level ˃ No MSMS were acquired for the 1pmol standard ˃ Evidence for n-terminal labeled peptides [M+H]2+

» Threshold for Survey MSMS = 30,000 counts

WF-A: In-Solution Labeling In-gel Digest

WF-B: In-gel Labeling In-gel Digest

1780 1800 1820 1840 1860 1880 1900 1920m/z

1843.673

1884.786

1853.728 1881.702

1851.8681901.047

1900.926

1794.755

1900.8931844.618

1797.756

755

75.715

1837.927

1792.363

1856.8291791.702

1835.687 1871.710 192

1867.683 1894.8051837.676

0 600 800 1000 1200 1400 1600 1800m/z

1128.538587.276

1029.469 1454.740

1454.7271128.518587.275782.483 1443.723

773.355

1029.478650.456 1241.653

1454.711

1241.635

1128.522

883.403

901.416

1811.012

1436.765

1436.699

587.228 1826.724

537.287

568.288 908.454 1596.877728.300

1443.7281240.523

1223.574

1813.769500.289

1595.808

1596.735

1 pmol

5 pmol

0.3 pmol

» Evidence of peptide [M+H]+ and MS/MS spectra » Detection of peptide at 0.3 pmol using MALDI/TOF

MS detection of intact peptides MS/MS spectra of m/z 1843.7

WF-B: In-gel Labeling In-gel Digest

1 pmol

5 pmol

0.3 pmol

» Evidence of peptide [M+H]+ and MS/MS spectra » Detection of peptide at 0.3 pmol using MALDI/TOF

MS/MS spectra of m/z 1843.7

b6 b7 b8 b10 b11 b13

In-Solution Labeling In-Solution Digest

» Post-solution labeling, addition of 4% ammonium hydroxide is essential to quench solution digest

Table 1. Dimethyl In-Solution Labeling Amount of Protein in Reaction

Reagent 5 pmol 1 pmol 0.3 pmol 1 pmol/uL protein in 100 mM Na acetate, pH 5 5 uL 1 uL 0.3 uL 100 mM Na acetate, pH 5 - 4 uL 4.7 uL 4% Formaldehyde 1 uL 1 uL 1 uL 260 mM Na cyanoborohydride 1 uL 1 uL 1 uL Reaction Conditions vortex 5 min RT vortex 5 min RT vortex 5 min RT 4% ammonium hydroxide (quench reaction) *optional, but required for in-solution digest

1 uL, vortex 1 uL, vortex 1 uL, vortex

Final Volume 8 uL 8 uL 8 uL

In-Solution Labeling In-Solution Digest

» Post-solution labeling, addition of 4% ammonium hydroxide is essential to quench solution digest

»

» Failure to quench may result in uncontrolled dimethylation of peptides during in-solution digest

Table 1. Dimethyl In-Solution Labeling Amount of Protein in Reaction

Reagent 5 pmol 1 pmol 0.3 pmol 1 pmol/uL protein in 100 mM Na acetate, pH 5 5 uL 1 uL 0.3 uL 100 mM Na acetate, pH 5 - 4 uL 4.7 uL 4% Formaldehyde 1 uL 1 uL 1 uL 260 mM Na cyanoborohydride 1 uL 1 uL 1 uL Reaction Conditions vortex 5 min RT vortex 5 min RT vortex 5 min RT 4% ammonium hydroxide (quench reaction) *optional, but required for in-solution digest

1 uL, vortex 1 uL, vortex 1 uL, vortex

Final Volume 8 uL 8 uL 8 uL

In-Solution Labeling In-gel Digest

» Ensure neutral pH: 1M TrisHCl, pH 8.3, is added to each tube until bromophenol blue color is achieved.

» After in-gel labeling, rinse gel pieces with 4% NH4OH ˃ Aids in quenching of dimethylation reactants

In-gel Labeling In-gel Digest

Q: “Cannot easily visualize bands for 1 and 0.3 pmol” A: Recommend Silver Stain for 0.3 pmol protein » Some labs excised the “invisible” gel band co-

migrating with the visible myoglobin in appearing at 1 and 5 pmol

15%T SDS-PAGE followed by Ag stain

5 5 1 1 0.3 0.3 Protein amount (pmol) MW Ladder

Lab 21R

Q: “Why did we lose myoglobin after in-solution labeling”?

A: Many participants reported major loss of protein » Some possibilities:

˃ Losses during de-salting of the intact protein ˃ Losses may occur if the reaction solution is not fully

neutralized before SDS-PAGE

» 6 participants identified Nt-dimethylated myoglobin as the miscleaved peptide spanning K16

» Many also identified the Ct fragment with internal lysine dimethylation

» All used Workflow A, in-solution derivatization

peptide native

[M+H]+ dimethyl [M+H]+

2x dimethyl [M+H]+

GLSDGEWQQVLNVWGK 1815.90 1843.93 1871.96

GLSDGEWQQVLNVWGKVEADIAGHGQEVLIR 3403.74 3431.77 3459.80

NDIAAKYKELGFQG (c-term fragment) 1553.79 1581.83 1609.86

Q: “In the tryptic peptide GLSDGEWQQVLNVWGK, why was the C-terminal Lysine not methylated?”

A: N-terminal of the intact protein is the primary site for Dimethylation reaction » Secondary reactions at the epsilon side chain of

Lysine also occur (minor) ˃ Dimethylated Lysines are miscleaved during in-gel

digestion ˃ Typically reported as internal, dimethylated Lysines

rather than C–terminal Lysines » e.g.: in ASEDLKKHGTVVLTALGGILK, internal Lysines

are often dimethylated.

» Workflow B approach performed on several projects from Memorial Sloan Kettering Cancer Center laboratories

» PIs volunteered gel-bound and PVDF-bound proteins ˃ LC-MS with dimethylation ˃ Comparative Edman degradation experiments

» In all instances, the majority of Lysine dimethylated peptides are found to reside in a miscleaved peptide.

» Chang et al., ABRF 2015 Poster # 145: N-terminal amino acid sequence determination of proteins by N-terminal dimethyl labeling; pitfalls and advantages when compared with Edman degradation sequence analysis

» LC-MS with vented column/trap loading helps remove excess reagent

» For in-solution labeling, manual desalting or FASP protocol improves sensitivity

» For database searching, include a net mass addition of 28.0313 (C2H4) to the N-terminus and lysine amino acids as a variable modification

» Search engines may force the data to fit N-terminal Dimethyl to peptides with internal lysines ˃ Matched y and b ions cannot distinguish between Nt

dimethyl and internal Lysine dimethylation

NDIAAQYKELGFQG ASEDLKKHGTVVLTALGGILKK ALELFRNDIAAQYKELGFQG YKELGFQG

No Lysine dimethylation is specified: search engines fit MS/MS to

non-specific Nt-dimethylation:

Lysine dimethyl used as a variable mod: we will obtain the following hits for

internal Lysine dimethylation:


» Search engines may force the data to fit N-terminal Dimethyl to peptides with internal lysines ˃ Matched y and b ions cannot distinguish between Nt

dimethyl and internal Lysine dimethylation


No Lysine dimethylation is specified: search engines fit MS/MS to

non-specific Nt-dimethylation:

Lysine dimethyl used as a variable mod: we will obtain the following hits for

internal Lysine dimethylation:

NDIAAQYKELGFQG ASEDLKKHGTVVLTALGGILKK ALELFRNDIAAQYKELGFQG YKELGFQG • Dimethylated lysines receive higher scores

• Must use Nt-dimethyl as well as Lysine dimethyl among the variable search parameters

Spectrum of Myoglobin dimethylated peptide: IPIKYLEFISDAIIHVAHSK

Where is the dimethyl group located?

“Observation of dimethylation at the N-terminus of I99”




• b1, b2, b3 not detected (below cutoff)

• y17, y18, y19 not detected (above cutoff)

• b4 contains +28 Da confirms dimethyl at Nt I1 OR at internal K4




• b1, b2, b3 not detected (below cutoff)

• y17, y18, y19 not detected (above cutoff)

• b4 contains +28 Da confirms dimethyl at Nt I1 OR at internal K4

Conclusion: MS/MS spectrum does not support the exact positioning of Dimethyl group

» After doing both 2014 and 2015 studies, I would choose TMPP labeling method over dimethylation because the TMPP reagent is less likely to hit epsilon lysine.

» Worst offender was probably the size of the N-terminal peptide at 3460 Da. The C-terminal peptide was half that size and was easily seen and fragmented. Perhaps I should have investigated using a different cleavage enzyme.

» An in-gel digest was performed and the 3460 Da mass was difficult to extract from the gel.

» Digestion was really poor even with good pH control. The sodium acetate was a real pain.

» With all attempts made the recoveries were very poor and hardly any peptide could be identified.

» Protocol and explanations should be more detailed. » You should deliver the FASTA file to process more objectively.

» This was fun!

» Sample prep is crucial ˃ Minimize loss of material ˃ Desalting/removal of excess reagent

» Tight control of reaction conditions is required ˃ pH is critical ˃ Adjustment of pH after reaction may affect efficiency of trypsin digest

» Manual inspection of data ˃ Verify that b-ions can support location of derivitization ˃ Dimethylation of internal lysines can complicate data interpretation

» Dimethyl labeling may be a useful tool for Proteomics Labs ˃ Independent of identity of Nt-Amino Acid, can be universally applied

» Updated protocol to be added to the PSRG/ABRF webpage

» See our other related posters: ˃ Chang et al., ABRF 2015 Poster # 145: N-terminal amino

acid sequence determination of proteins by N-terminal dimethyl labeling; pitfalls and advantages when compared with Edman degradation sequence analysis

˃ ABRF PSRG Poster # 260: N-terminal identification of a standard protein at low picomole levels by dimethyl labeling and bottom-up mass spectrometry

» We are always looking for new PSRG members!

» If you have interest in protein sequencing, and skills with either Edman or mass spectrometry, please contact one of our current members

Robert English Shimadzu Scientific

[email protected]

Sara McGrath FDA/CFSAN

[email protected]

» Sponsors of study proteins and reagents:

ABRF

Biomedical Research Core Facilities University of Michigan

» Anonymizer:

Amanda McGinnis, University of Michigan

» ………and study participants!!!!!!

» Sara McGrath (co-chair) FDA/CFSAN » Robert English (co-chair) Shimadzu » Greg Cavey Southwest Michigan

Innovation Center » Hediye Erdjument-Bromage MSKCC/Rockefeller

Research Laboratories » Mark Garfield NIH/NIAID » Xuemei Luo Univ. of Texas Medical

Branch » Henriette Remmer Univ. of Michigan » Brett Phinney (EB liaison) UC Davis

» Orthoproteogenomics: Multiple proteomes investigation through orthology and a new MS based protocol, S Gallien, E Perrodou, C Carapito, C Deshayes, JM Reyrat, A Van Dorsselaer, O Poch, C Schaeffer, O Lecompte, Genome, 19, 128-135 (2009)

» Stable-isotope dimethyl labeling for quantitative proteomics. JL

Hsu, SY Huang, NH Chos, SH Chen Analytical Chemistry, 75, 24, 6843-52. (2003)

» In-gel digestion for mass spectrometric characterization of

proteins and proteomes. Andrej Shevchenko, Henrik Tomas, Jan Havlis, Jesper V Olsen & Matthias Mann, Nature Protocols, 1, 6, p 2856-2860 (2006)

» Universal sample preparation method for proteome analysis.

Jacek R Wiśniewski, Alexandre Zougman, Nagarjuna Nagaraj & Matthias Mann, Nature Methods 6, 359-362 (2009)

Protein Sequencing Research Group ABRF 2015 annual meeting · Protein Sequencing Research Group ....

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