Page 20 - Pharmaceutical- Guide to Biopharmaceutical

Characterization Quality Control

Protein Primary Structure Analysis PPSQ-51A / 53A

N-Terminal Amino Acid Sequencing of Mouse benefits

IgG Using PPSQ-51A/53A Gradient System Cell Line Optimization
click here • Amino acids can be sequenced accurately with extremely high data reliability.
• Ile and Leu residues with identical masses can be differentiated and the presence and
Operating Principle and Features IgG derived from mouse position of S-S bonds can also be determined.
serum 2 pmol
The PPSQ protein sequencer automates the Edman degradation • Proteins can be analyzed directly, which makes operations extremely easy.
process.
Although using Edman degradation to determine amino acid SDS-PAGE (Gel concentration Culture
sequences is very time-consuming, the reliability of the resulting amino 4-12 % Bis-Tris gel)
acid sequences is very high, making it especially useful for protein
amino acid sequencing when no database has been built. PTH-amino H-chains
acids obtained by Edman degradation are analyzed by isocratic or Electroblotting (tank type)

gradient elution. on a PVDF membrane L-chains
Measurement Method and Conditions
CBB staining, destaining
To operate the sequencer, proteins or peptides to be analyzed are
applied to a glass filter treated with polybrene or transferred to a PVDF Purification
membrane. After electrophoresis, they are stained and placed in the Amino acid sequencing
reactor with an excised protein spot. After that, they can be analyzed using the PPSQ
automatically. In this example, samples were prepared by reducing 2 Fig. 1 Protocol for Analysis Fig. 2 PVDF Membrane after Electroblotting
pmol of IgG from mouse serum, separating that into H and L-chains by
SDS-PAGE, transferring the chains onto a PVDF membrane, staining,
destaining, and then excising the resulting bands (Fig. 1 and 2). The Table 1 Analysis Conditions (Gradient System)
IgG is reduced and separated into H and L-chains. The H and L-chains Column: Wakopak Wakosil PTH-GR
were separated and purified based on conditions indicated in Table 1 (S-PSQ, 250 mm×2.0 mm I.D.)
and then their amino acid sequences were analyzed (Fig. 3). Mobile phase A: PTH-amino Acids Mobile Phase A
(for Gradient Elution)
Mobile phase B: PTH-amino Acids Mobile Phase B
Results (for Gradient Elution) Characterization
Flowrate: 0.3 mL/min PPSQ-51A/53A Isocratic System
The amino acids in L-chains can be identified to 13 residues from Column Temp.: 35 °C
the N-terminal, as Asp-Ile-Gln-Met-Thr-Gln-Ser-Pro-Ala-Ser-Leu-Ser- Detection: SPD-M30A (269 nm)
Ala(Val). A database search confirms that the sequence is for an Flow Cell: High Sensitivity Flow cell Specifications
immunoglobulin kappa light chain (Fig. 2).
Instrument PPSQ-51A PPSQ-53A
Reaction method Edman degradation
Reaction time 46.5 min/cycle 48 min/cycle Quality Control
Number of reactors 1 3
Cycle 1 Sample retention method 8 mm diameter glass fiber disc or PVDF membrane
Reactor temperature control range Room temp. +10 to 60 °C
Cycle 2 Converter temperature control range Room temp. +10 to 70 °C
Cycle 3
Number of samples/solvents 7
Cycle 4
Sample/solvent supply method N2 gas pressure
Cycle 5 Dimensions W 510 mm × D 500 mm × H 540 mm
Weight 43 kg 45 kg Pharmacokinetics

Fig. 3 Chromatogram of L-Chain (Raw Chromatogram from First Cycle and Difference Chromatograms from Cycles 2 to 5) Elution method Isocratic or gradient system
Mobile phase Special eluent specifically for Shimadzu protein fully-automated protein sequencers*
Conclusion Application Examples
Reaction reagent Reagent specifically for Shimadzu protein fully-automated protein sequencers*
PPSQ-51A/53A systems can determine N-terminal sequences easily and • Identifying the primary sequence of peptides Column Column specifically for amino acid sequencing*
accurately. The gradient system can detect peaks with approx. 3 to • Identifying the presence and position of S-S bonds Others
5 times higher overall peak height than when using isocratic elution, • Identifying post-translational modifications Power requirement Single-phase 120-230 V AC, 50/60 Hz, 1,500 VA max
which means amino acid sequences can be determined for even trace Nitrogen gas Min. 99.9999 % purity
quantities of protein.
* Available for purchase from Fujifilm Wako Pure Chemical Corporation.

20 21
index index