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Rapid Peptide Mapping via Automated Integration of On-line Digestion, Separation and Mass Spectrometry for the Analysis of Therapeutic Proteins
Overview
Purpose:
An automated system was developed that integrated rapid trypsin digestion
without pretreatment, on-line desalting and high resolution LC/MS/MS.
Methods:
A rapid sample preparation and separation system equipped trypsin column
was used for fast trypsin digestion of reduced as well as native proteins. This sample
preparation instrument was coupled to a hybrid quadrupole-Orbitrap mass
spectrometer for peptide mapping experiments and the resulting data sets were
analyzed.
Results:
For the reduced and alkylated antibody sample, a 100% sequence coverage
of the light chain and 91% sequence coverage of the heavy chain were observed. For
the untreated/native antibody sample, 90% sequence coverage of the light chain and
73% sequence coverage of the heavy chain were observed. Furthermore, a digest of
native serum albumin resulted in 51% sequence coverage. The low coverage
percentage value associated with the untreated/native protein analytes can be
attributed to disulfide bonded peptides that were likely recovered from the analytical
system but not recognized by the proteomics software
.
Introduction
Biological systems are extraordinarily dynamic. As such, it is often the case that the
time associated with protein sample preparation and analysis delays the detection of
malformations until it is too late to take action. Furthermore, a lack of hands-free
automation puts an enormous strain on analysts to replicate results across multiple
labs. Presented here, is the automated integration of rapid sample preparation,
separation and MS used for peptide mapping of a reductively alkylated monoclonal
antibody, untreated (native) antibody and untreated human serum albumin (1, 2).
Methods
Samples
To reduce the intact mAb, the sample was incubated for 1 hour at 60 C in 6 M
guanidine HCl containing 25 mM DTT for complete reduction. Following reduction the
samples was reacted with 100 mM iodoacetic acid for 1 hour at room temperature.
Native samples were simply diluted to their final concentration in tris-buffered saline pH
7.4 prior to injection. Peptide mapping of all samples was performed using a modified
Dionex ultimate 3000 RSLC nano system system equipped with a Perfinity No
Reduction or Alkylation (NORA) Trypsin Column, Digest Buffers from Perfinity, a C18
trap column (Halo, 0.3 X 20 mm) and C18 analytical column (Halo 0.3 X 100 mm).
Online Trypsin Digestion
Untreated antibody solutions were directly injected into the system. Digest efficiency
was monitored at various times and temperatures. Simultaneous denaturation and
enzymatic digestion were performed at 70 C. Reductively alkylated samples were
processed at 50 C. 2 minute digestion times were utilized for all samples.
Chromatography
A Dionex Ultimate 3000 RSLC nano system equipped with a microflow flow selector
pumping at 20-50 uL/min was used for the desalting and reversed phase operations.
Following digestion, the trapping column was brought in-line with the analytical column
by valve switching. Both columns were desalted for 3 column volumes using initial
gradient conditions. For peptide mapping experiments, peptides were eluted with a 50
min gradient at a flow rate of 20
µ
L/min. LC solvents are 0.1% formic acid in H
2
O
(Solvent A) and 0.1% formic acid in acetonitrile (Solvent B). LC gradient was 0-50%B in
50 min.
Figure 1 shows the schematics of the LC configuration used for automated peptide
mapping. All transfer tubing was 75 µm Thermo Scientific™ Dionex™ nanoViper™
fingertight fitting system. All valves and columns were operated under isothermal
condition in a column oven. An isocratic pumping system was utilized for all sample
preparation steps while binary pumps were used for the reversed phase gradient. This
configuration enables the digestion of a sample while another sample is separated by
reversed phase. By parallel processing in this way a sample can be run every 7
minutes.
Mass Spectrometry
Peptides eluted from analytical column were analyzed using a data-dependent top 10
experiment on the Thermo Scientific
TM
Q Exactive
TM
hybrid quadrupole-Orbitrap mass
spectrometer (Figure 2). Resolution was 70K for the full MS, 17.5K for HCD MS/MS
with a dynamic exclusion of 30 seconds. Detailed instrument parameters are listed in
Table 1.
FIGURE 1. Schematics o
Figure 2. Schematic of t
spectrometer
.
HCD Cell, C-Trap for
Spectrum Multiplexing
Orbitrap with
Advanced Data Processi
Table 1. The Q Exactive
instrument method para
Thermo Scientific
TM
Prote
protein database with the
disulfide linked peptides w
Exactive hybrid quadrupol
10 ppm precursor mass tol
