c190-e161a perfinity idp - shimadzu
TRANSCRIPT
C190-E161A
Integrated Protein Digestion HPLC
Perfinity iDP
Manual Method
DigestionProteins are rapidly digested by trypsin immobilized onto the stationary phase of the column.
DesaltingTryptic peptides are concentrated and desalted on the desalting column.
Perfinity iDP, a new platform for protein analysis, automates the protein digestion process
Automated Digestion Workflow
The Perfinity iDP (Integrated Digestion Platform) automates the protein analysis workflow from protein digestion to HPLC separation
and MS detection, significantly reducing sample preparation times and enhancing reproducibility.
Protein
To reversed phase separation
Trypsin
Reduce trypsin digestion time to
1-4 minutes
HPLC or LC/MSanalysis
Re-suspension
Solid phase
extractionProtein digestion 18-24 hours
Peptide
Peptide
Desalting columnTrypsin column
Perfinity iDP
Perfinity iDP reduces the entire sample preparation
workflow down to 20 minutes
Protein sample
Desalting column 2
DetectorReverse phase column
Desalting column 1Trypsin column
The protein sample is delivered into the trypsin column by optimized digestion buffer.
Sample can be multi-plexed using two desalting column to increase throughput.
HPLC or LC/MS analysis
Protein digestion
(1-4minutes)Desalting
2
Features
Perfinity and Perfinity iDP are registered trademarks of Perfinity Bioscience, Inc.
Perfinity iDPIntegrated Protein Digestion HPLC 3
10.0 12.5 15.0 17.5 20.0 22.5 25.0 min
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0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5min
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Fast On-Line Trypsin Digestion
1-4 minutes trypsin digestion using a high-efficiency trypsin column
The benefits of using Perfinity iDP with the immobilized
trypsin column are:
1. Fast trypsin digestion in 1 to 4 minutes
2. Continuous sample analysis on reusable trypsin
column
3. Reduced chymotrypsin activity and deamidation
Insulin was digested within 1 minute using the trypsin
column as shown in the figure at right.
* Optimal digestion time depends on proteins.
Optimized Perfinity iDP digestion buffer
The optimized trypsin digestion buffer in combination
with the trypsin column enables highly efficient
digestion.
The figure to the right shows a comparison between the
optimized digestion buffer and the commonly used tris
buffer. The optimized digestion buffer provided a higher
peptide recovery rate with a short digestion time.
Perfinity iDP trypsin buffer
Tris buffer
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5min
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Increased throughput by parallel sample processing withtwo desalting columns
Two desalting columns allow parallel processing of
samples. One desalting column is used to trap digested
peptides, while peptides from the previously injected
sample are eluted from the other desalting column.
This parallel sample processing allows analysis of up to
200 samples/day.
The figure to the right is an example of parallel sample
processing, which increases throughput while
maintaining reproducibility. Parallel process by two desalting column
Increased digestion efficiency by the Perfinity iDP digestion buffer
Digestion of insulin (5 μg)
Insulin
Digested insulin
Digestion time : 15 sec
Digestion time : 30 sec
Digestion time : 1 min
4
The Perfinity iDP software supports the total workflow from method development to analysis of samples
Easy Operation by Per�nity iDP Software
Method settingsAfter selecting a method, users can
quickly create and optimize methods
by entering a few parameters, such as
digestion time, column dimensions,
and gradient time. Per�nity iDP
methods are then automatically
generated for running samples.
Batch file creationBatch �les for sample analysis are
quickly generated. Users enter minimal
information such as methods, data �le
names, and injection volumes.
Seamless connection with LabSolutionsAnalysis results can be viewed in the
LabSolutions post-run window, which
can be accessed through the Per�nity
iDP software.
Step
1
Step
2
Step
3
Perfinity iDPIntegrated Protein Digestion HPLC 5
20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 min
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uV
15.0 20.0 25.0 30.0 35.0 40.0 min
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Full Automation Enables Reliable Analysis
Increased reproducibility by automation of workflow
Perfinity iDP minimizes human error by automating all
steps from digestion to desalting to LC/MS analysis.
The figure at right shows reproducible chromatograms of
reduced and alkylated transferrin analysis.
Minimized carryover
Reliable instrumentation and optimized methods
result in very low levels of carryover.
The figure at right shows a blank injection after
a transferrin injection to illustrate minimized
carryover with reliable results using Perfinity iDP.
Reproducibility of 20 peptides that was produced by digestion of reductive alkylated transferrin
Time20
20.420.921.121.522.222.422.722.923.223.423.523.623.824
24.224.424.825
25.5
CV(%)5.247.73.631.026.070.442.460.664.873.542.435.251.391.856.857.792.351.851.796.63
StDev4014902038161699148121275446194753529125311626344431914361466501981454484802262
Average76602.33117181.7105172.3166716244098289735
181448.3143593109811
257738.3128491.750219.33
31791103218.352781.3385325.3384397.67245547
474045.734121.33
Run 380057121037105673167296234215288299178861142517110346249757129993491503146710182453091781488214925066646736731764
Run 272199106875108713168049236951290735178884144298114875255772124909482883229610540156231865498588324398347118436274
Run 177551123633101131164803261128290171186600143964104212267686130573532203161010243049022912798516124199248358634326
Analysis of reduced and alkylated transferrin
Carryover of transferrin
6
Wide application range with LC/MS
Application : Detection of IgG digested peptides by LCMS-2020
IgG was analyzed Perfinity iDP with LCMS-2020, Main peptide fragments were easily confirmed after 4 minutes digestion.
MS spectrum of peak 1 (retention time : 10.01 min) MS spectrum of peak 3 (retention time : 30.25 min)
MS spectrum of peak 2 (retention time : 17.30 min)
MS (TIC) chromatogram of IgG (300 pmol)
LC chromatogram (UV 214 nm)
Perfinity iDPIntegrated Protein Digestion HPLC 7
Perfinity iDP
Printed in Japan 3655-08313-20AIT
Company names, product/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation or its affiliates, whether or not they are used with trademark symbol “TM” or “®”.Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services. Shimadzu disclaims any proprietary interest in trademarks and trade names other than its own.
For Research Use Only. Not for use in diagnostic procedures. The contents of this publication are provided to you “as is” without warranty of any kind, and are subject to change without notice. Shimadzu does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication.
© Shimadzu Corporation, 2013www.shimadzu.com/an/
CBM-20A
Reservoir tray
DGU-20A5R
LC-20ADXR
LC-20AD
LPGE unit
Rinse kit for 20AD
SIL-20ACHT UFLC
Mixer MR 100 μL
CTO-20AC
SPD-20A
FCV-36AH
Startup kit
System configuration
1
2
1
2
1
1
1
1
1
1
1
2
1
Item Quantity
Trypsin column
Mobile phase kit
Pretreatment kit
228-56951-10
228-56951-60
228-56951-64
Columns and Consumables
Item
Piping kit
Data process
Perfinity iDP software
Manual
Perfinity and Perfinity iDP are registered trademarks of Perfinity Biosciences, Inc.
Windows is a registered trademark of Microsoft Corporation.
LabSolutions is a registered trademark of Shimadzu Corporation.
Startup kit
Item
P/N
1
1
1
1
Quantity
Perfinity iDP software and a dongle
Remarks