N - and C-Terminal Protein Sequencing by MALDI ISD Feb 2009

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Application note: N- and C-terminal sequencing by MALDI ISD # 2000902

N- and C-terminal Protein Sequencing


Using in-source-decay (ISD) MALDI MS

By Ejvind Mortz, Thanh Ha Nguyen, and Janne Crawford. Alphalyse A/S, Unsbjergvej 4, Odense, Denmark.
Email correspondence: [email protected]

Analytical characterization of purified proteins, in particular recombinant proteins for drug discovery and development, requires confirmation
of the full length protein sequence. Here we present a powerful mass spectrometry method (MALDI ISD) that provides partial sequence
information of the intact protein with up to 20-50 amino acid residues from both the N-terminal and C-terminal in one single analysis.
The analysis can thus confirm expression and purification of the full length protein sequence, and detect unexpected truncations and
modifications of the termini. The MALDI ISD analysis can also be applied to N-terminally blocked and PEGylated proteins. Alphalyse
provides protein analysis services and here we present results obtained on a variety of proteins.

INTRODUCTION
Structural characterization of purified natural and down sequencing of peptides and intact proteins (refs 1-5).
recombinant proteins traditionally includes N-terminal
In this article, Alphalyse presents results obtained using
Edman sequencing to confirm 5-15 amino acid residues from
ISD MALDI mass spectrometry on a range of purified
the amino terminus. N-terminal sequencing is a requirment
proteins with Mw from 6-80 kDa. MALDI ISD offers
according to the ICH Q6B Guideline for characterization of
several key benefits compared to Edman sequencing:
recombinant proteins for clinical testing, and to demonstrate
comparability and consistency between cGMP batches. • Both N- and C-terminal sequences of 20-50 residues
N-terminal Edman sequencing does not work for can be obtained.
N-terminally blocked proteins, and the analysis is time- • N-terminally modified and blocked proteins (acetyla
consuming with a cycle time of 40 minutes per amino acid. ted, pyroglutamate, PEGylated) can be sequenced.
An alternative technique, ISD MALDI MS has been • Data acquisition is fast.
developed and demonstrated in several publications for top- • Very long sequence reads can be obtained with up to
80 residues from one single ISD MALDI mass
spectrum.

FIGURE 1. PRINCIPLES OF ISD MALDI MS


The intact protein is fragmented in the MALDI ion source. The
reflector MALDI spectrum shows intense c-ions (and a-ions)
for N-terminal sequencing, as well as y-ions (and z+2 ions) for
C-terminal sequencing. Reflector MALDI spectra from 700-
5000 Da typically cover 7-50 residues from the terminals.

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Application note: N- and C-terminal sequencing by MALDI ISD # 2000902

MATERIALS AND METHODS


Purified proteins were obtained from collaborators and from to yield predominantly c-type fragments with high
various commercial suppliers. Two samples were obtained abundance, and y-, z- and a-ions at lower abundance. The
from the ABRF-ESRG 2009 research study. Proteins were mass spectra were calibrated using external calibration
reduced with DTT and alkylated with iodoacetamide, with a standard peptide calibration mixture.
and subsequently purified using Biomax-10 Spinfilters or The MS data were correlated to the protein sequences
Ziptips (Millipore). The protein samples (20-50 pmol) were by Mascot database searching (Matrix Science). The
deposited on a stainless steel target and co-crystallized Sequence Editor tool in BioTools (Bruker Daltronics) was
with a matrix of 2,5-DHB, 1,5-DAN, or sinapinic acid used for de-novo sequencing and detection of sequence
(refs 1-5). The MALDI mass spectra were acquired on an modifications.
Autoflex III instrument (Bruker Daltronics) in ISD-mode
where the protein ions spontaneously fragment

RESULTS
A range of different proteins with molecular weights from residues. Only 10 amino acids in the middle of the small
6.5 to 77 kDa were analyzed by MALDI ISD. The smallest protein were not covered by the analysis. Thus, 83% of the
protein, the aprotinin peptide of 6.5 Da (Figure 2) showed 2 sequence is confirmed in one single MS analysis.
ion-series that confirmed 25 N-terminal and 23 C-terminal

FIGURE 2 ISD MALDI SPECTRUM OF APROTININ (6.5 kDa)


The MALDI ISD spectrum was used for Mascot database searches (insert) that identified the protein. A c-ion series confirms
25 N-terminal amino acids, and a y-ion series confirms 23 C-terminal residues. The ions and sequences are annotated in the
spectrum.

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Application note: N- and C-terminal sequencing by MALDI ISD # 2000902

The Association of Biomolecular Resource Facilities spectrum covered 30 N-terminal and 29 C-terminal amino
(ABRF) 2009 study for comparing Edman and MS based acid residues. The protein in Sample 2 was identified as
techniques for N-terminal analysis included two protein GAPDH (P46406).
samples provided to a broad range of laboratories as a The MALDI ISD spectrum shown in Figure 3 confirmed
blinded study. The 2 protein samples were analyzed by 39 N-terminal and 32 C-terminal residues. The leading
MALDI ISD. The protein in Sample 1 was identified as a Methionine residue in the database sequence was not
fusion protein between a his-tag expression vector and present in the N-terminal. See reference 6 for the complete
alcohol dehydrogenase (P00330). The correct protein was ABRF-ESRG 2009 study results for comparison of Edman
identified by a combination of Mascot database searching and MS techniques for N-terminal sequencing.
and de-novo sequencing of the fusion area. The MALDI ISD

FIGURE 3 ABRF - ESRG STUDY. ANNOTATED ISD MALDI SPECTRUM OF SAMPLE 2 (36 kDa)
The ISD MALDI spectrum was used for a Mascot database search (insert) in the NCBI nrdb database for identification of the
protein. The spectrum confirms both the N- and C-terminals of the database sequence, covering in total 71 amino acid residues
in a single mass spectrum.

In collaboration with ACE BioSciences A/S, a recombinant MALDI ISD analysis confirmed the expected C-terminal,
vaccine protein was analyzed by MALDI ISD (Figure 4). The and covered 43 N-terminal and 37 C-terminal residues in
Mascot database search confirmed an expected N-terminal total.
truncation where a leader sequence is removed. The

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Application note: N- and C-terminal sequencing by MALDI ISD # 2000902

FIGURE 4. ISD MALDI SPECTRUM OF RECOMBINANT VACINE PROTEIN (21 kDa)


The ISD MALDI spectrum and Mascot database search (insert) confirms the C-terminal of the database sequence, and
demonstrates an N-terminal truncation. The analysis confirms in total 80 residues in a single ISD MALDI spectrum.

The range of different proteins analyzed by MALDI ISD lengths from 19-43 residues. In several proteins N-terminal
for this application note is summarized below in Table truncations were detected. For the monoclonal antibody,
1. The analysis was successful for proteins from 6 to the analysis confirmed the expected N- and C-terminals
80 kDa. For all proteins analyzed both N-terminal and for both the heavy and the light chains. An N-terminal
C-terminal sequences were obtained with sequence pyroglutamate residue was detected in the heavy chain.

TABLE 1. LIST OF PROTEINS ANALYZED BY MALDI ISD

PROTEIN NAME MW [kDa] N-TERM C-TERM COMMENTS

Aprotinin 6.5 25 AA 23 AA

Myoglobin 16.9 33 AA 31 AA

ACE393 18.8 43 AA 37 AA N-terminal truncation

GAPDH 35.8 21 AA 24 AA

BSA 69.3 33 AA 27 AA N-terminal truncation

Transferrin 76.9 43 AA 19 AA

ABRF study 2009: His-tagged ADH 40.1 30 AA 29 AA

ABRF study 2009: GAPDH 35.8 39 AA 32 AA N-terminal Met absent

Anti hOX40 light chain 24.2 34 AA 29 AA

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Application note: N- and C-terminal sequencing by MALDI ISD # 2000902

CONCLUSION
In the present study, the MALDI ISD technique showed • MALDI ISD worked efficiently for all analyzed proteins
several advantages compared to traditional Edman from 6-80 kDa.
sequencing. A comparison of the 2 techniques is given in • Both N-terminal and C-terminal sequences were
obtained for all proteins.
Table 2. The features and advantages of MALDI ISD make
it a very useful technique for analysis and quality control of • Long sequence reads of 19-43 residues were obtained
from both termini.
purified natural and recombinant proteins.
• Truncated and N-terminally blocked proteins could be
sequenced.

TABLE 2. COMPARISON OF MALDI ISD AND EDMAN TECHNIQUE FOR PROTEIN SEQUENCING

TECHNIQUE EDMAN MALDI ISD

N-terminal sequence Yes Yes

C-terminal sequence No Yes

Sequence lengths 5-50 residues 20-50 residues

Sample requirements Purified protein. Purified protein

SDS PAGE purified protein on PVDF membrane

Disadvantage • Low throughput analysis with cycle time 40 • Due to MALDI matrix ions, the first 5-7 amino
mins per residue. acids can only be sequenced directly on
• Expensive chemicals results in high cost per instruments with “T3-sequencing” utility.
residue. • De-novo sequencing of novel proteins is
• Does not work for N-terminally blocked difficult.
proteins. • Isobaric amino acids cannot be distinguished
• No information about the C-terminal (I/L, Q/K).

Advantage • Can be combined with 1D and 2D • C-terminal sequencing possible.


electrophoresis for analysis of proteins in • Rapid analysis.
mixtures. • Modified proteins and N-terminally blocked
• True sequencing of individual aa makes de- proteins can be sequenced.
novo sequencing possible.
• Modified proteins and N-terminally blocked
proteins can be sequenced.

REFERENCES
1. Reiber, DC et al. Anal. Chem.. 1998, 70, 673-683. Identifying proteins using matrix- assisted laser desorption/ionization in-source fragmentation data
combined with database searching.
2. Takayama, M et al. Electrophoresis 2000, 21, 1670-1677. Sequence information of peptides and proteins with in-source decay in matrix assisted laser
desorption/ionization-time of flight mass spectrometry.
3. Schnaible, V et al. Anal. Chem. 2002, 74, 4980-4988. Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS.
4. Demeure, K et al. Anal. Chem. 2007, 79, 8678-85. Rational selection of the optimum MALDI matrix for top-down proteomics by in-source decay.
5. Suckau, D et al. Bruker Daltronics Application Notes #MT-57. Reflector in-source-decay (ISD) MALDI-TOF MS: A powerful tool for N-terminal sequence
characterization of proteins.
6. Thoma, RS et al. The ABRF ESRG 2009 Study: Comparison of Edman and mass spectrometry techniques for N-terminal sequencing. http://www.abrf.
org/ResearchGroups/EdmanSequencing/EPosters/ESRG2009_poster(2009_02_03).pdf.

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