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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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

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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

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2

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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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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