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
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
K H3C CH3
» Nt-dimethyl peptides are best sequenced from b ions » Internal dimethylations can confound results
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
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:
NDIAAQYKELGFQG ASEDLKKHGTVVLTALGGILKK ALELFRNDIAAQYKELGFQG YKELGFQG
» 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:
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”
“Observation of dimethylation at the N-terminus of I99”
Spectrum of Myoglobin dimethylated peptide: IPIKYLEFISDAIIHVAHSK
Where is the dimethyl group located?
• 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
“Observation of dimethylation at the N-terminus of I99”
Spectrum of Myoglobin dimethylated peptide: IPIKYLEFISDAIIHVAHSK
Where is the dimethyl group located?
• 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
Sara McGrath FDA/CFSAN
» 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)