2-D hydrophilic interaction liquid chromatography-RP separation in urinary proteomics--minimizing variability through improved downstream workflow compatibility.

TLDR: The optimized and "streamlined" complex method has shown potential for use in future urinary proteomic studies and was tested in an extensive proteomic experiment on a kidney-transplanted patient.

Abstract: Optimization of every step in a bottom-up urinary proteomics approach was studied with respect to maximize the protein recovery and making the downstream steps in the workflow fully compatible without compromising on the amount of information obtained. Sample enrichment and desalting using centrifugal filtration (5 kDa cut-off) yielded protein recoveries up to 97% when 8 M urea was used. Although yielding lower recoveries (88%), addition of Tris-HCl/NaCl was considered a better choice due to good down-stream compatibility. The consecutive depletion of HSA, using an immunoaffinity column was successfully adapted for use in urine. Separation of the trypsin generated peptides in an off-line 2-D chromatographic system consisting of a hydrophilic interaction liquid chromatography column, followed by a RP chromatography column showed a high peak capacity and good repeatability in addition to a high degree of orthogonality. All operations were modified in order to keep sample handling between every step to a minimum, reducing the variability of each process. In order to test the suitability of the full method in an extensive proteomic experiment, a urine sample from a kidney-transplanted patient was analyzed (n=6). The total variability of the method was identified with RSD values ranging from 11 to 30%. Eventually, we identified a total of 1668 peptides and 438 proteins from a single urine sample despite the use of low-resolution MS/MS equipment. The optimized and “streamlined” complex method has shown potential for use in future urinary proteomic studies.

Figures

Figure 24. Fold change (log2) of immune proteins, growth factors and MEP1A in the rejection group, AR1 (●) AR2 (×) AR3 (♦) AR4 (+) AR5 (■) AR6 (▲), from baseline to Biopsy Proven Acute Rejection (BPAR). The center point (Clinically stable) is 7-11 days before BPAR, at stable serum creatinine levels.

Table 4. Different platforms used for peptide/protein identification

Figure 7. Illustration of 18O-incorporation by two different mechanisms, amide bond cleavage and carboxyl oxygen exchange (From reference [68] with permission).

Figure 8. Schematic workflow for peptide/protein identification after LC-MS/MS

Table 2. Overview of key variables and variability in different steps of the workflow

Figure 23. Scheme of the samples analyzed and compared from each patient

Full text

Searching for biomarkers of acute rejection in
renal transplant recipients – development and
optimization of a urinary proteomic approach
Thesis for the degree of Philosophiae Doctor
by
Håvard Loftheim
Department of Pharmaceutical Chemistry and Department of Pharmaceutical
Biosciences
School of Pharmacy
Faculty of Mathematics and Natural Sciences
University of Oslo
Norway

© Håvard Loftheim, 2011
Series of dissertations submitted to the
Faculty of Mathematics and Natural Sciences, University of Oslo
No. 1108
ISSN 1501-7710
All rights reserved. No part of this publication may be
reproduced or transmitted, in any form or by any means, without permission.
Cover: Inger Sandved Anfinsen.
Printed in Norway: AIT Oslo AS.
Produced in co-operation with Unipub.
The thesis is produced by Unipub merely in connection with the
thesis defence. Kindly direct all inquiries regarding the thesis to the copyright
holder or the unit which grants the doctorate.

TABLE OF CONTENTS
ACKNOWLEDGEMENTS
LIST OF PAPERS
ABSTRACT
LIST OF ABBREVIATIONS
1
Introduction ...................................................................................................................... 1
1.1 Kidney transplantation ............................................................................................. 2
1.1.1 Kidney transplantation in general and the status in Norway ..................................... 2
1.1.2 Acute rejections ................................................................................................. 3
1.2 Proteomics .................................................................................................................. 4
1.2.1 Sample preparation in urinary proteomics.............................................................. 5
1.2.2 Proteolytic digestion of proteins ........................................................................... 6
1.2.3 LC-MS/MS of proteins/peptides ........................................................................... 7
1.2.4 Quantification in urinary proteomics ................................................................... 12
1.2.5 Data acquisition ............................................................................................... 15
2 Aim of the study .............................................................................................................. 18
3 Results and discussion .................................................................................................... 19
3.1 Sample preparation and separation in urinary proteomics ................................ 19
3.1.1 Sample collection and storage ............................................................................ 20
3.1.2 Sample preparation ........................................................................................... 20
3.1.3 Chromatographic separation of the peptides ......................................................... 23
3.1.4 Variability of the method: step by step evaluation of the workflow ......................... 27
3.2 Tryptic digestion & protein identification ............................................................ 28
3.2.1 Optimization of digestion conditions using immobilized trypsin beads .................... 28
3.2.2 In-solution digestion vs. digestion on immobilized trypsin beads ............................ 29
3.2.3 Digestion efficiency in human urine ................................................................... 30
3.2.4 On-column reduction, alkylation and tryptic digestion .......................................... 31

3.2.5 Protein identification by different analytical platforms .......................................... 32
3.3 Accelerated quantification in urinary proteomics utilizing
18
O-labeling ........... 33
3.3.1 pH dependency and reaction time optimization .................................................... 34
3.3.2 Integration of digestion and labeling using immobilized trypsin beads .................... 36
3.3.3 Efficiency of the optimized procedure in urine samples ......................................... 39
3.4 Differential expressed proteins following acute rejection in renal transplant
recipients ............................................................................................................................. 42
3.4.1 Choice of patients and samples .......................................................................... 42
3.4.2 Up-regulated proteins ....................................................................................... 43
3.4.3 Comparison with earlier published data ............................................................... 51
3.5 Future perspectives ................................................................................................. 51
4 Concluding remarks ....................................................................................................... 53
5 References .................................................................................................................... .... 55

ACKNOWLEDGEMENTS
The presented work was performed at the department of Pharmaceutical Chemistry and the
department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo in co-
operation with both the department of Transplant Medicine at Oslo University Hospital and
the department of Chemistry, University of Oslo.
First of all I would like to thank my two supervisors Professor Léon Reubsaet and Professor
Anders Åsberg. I am very grateful for the opportunity to work under your guidance in the
borderline between two exciting research fields. Thank you for your great support and
enthusiasm; during these four years I have always been at my most inspired after having
meetings with you, seeing new opportunities and eager to test our new ideas in the laboratory.
I would like to thank my co-authors Thien Nguyen, Bjørn Winther, Bao Tran, Helle Malerød,
Elsa Lundanes, Tyge Greibrokk, Jadranka Vukovic, Karsten Midtvedt, Anders Hartmann,
Anna Varberg Reisæter, Pål Falck, Hallvard Holdaas and Trond Jenssen for your valuable
contribution to the work. A special thanks to my master students Thien, Malin and Tam; you
have made important contributions to my research.
I would also like to thank my colleagues for creating a great social working environment.
Your company has been much appreciated whether it has been in the laboratory or at
congresses and department trips.
Finally a warm thank you goes to my lovely wife Ragna for all the support you have given
me during this work. Spending time with you and our wonderful children Mari and Sverre
will always be the highlight of the day. You always make me smile when I come home
regardless of how bad the mass spectrometer has treated me during the day.
Oslo, August 2011
Håvard Loftheim

Frequently asked questions

Q1. What have the authors contributed in "Searching for biomarkers of acute rejection in renal transplant recipients – development and optimization of a urinary proteomic approach" ?

The main steps were desalting/enrichment by cut-off centrifugation ( 5 kDa ), albumin depletion and tryptic digestion followed by 2D-LC-MS. In Paper II enzymatic digestion using immobilized trypsin beads was investigated. In Paper III a multidimensional on-line system including Strong Anion Exchange Chromatography ( SAX ) separation of native proteins, reduction, alkylation, C4 separation and tryptic digestion of the alkylated proteins followed by MS detection was tested as an alternative to the off-line method developed. In Paper IV proteolytic O-labeling of peptides was investigated and improved in order to optimize the labeling efficiency and accelerate the process. On-line tryptic digestion was satisfactory for several proteins but needs further optimization to cover the full proteome. The system was evaluated using both model proteins and human urine sample and has shown potential as a tool to identify biomarkers offering short analysis time and minimum manual sample handling. 

Q2. What are the future works in "Searching for biomarkers of acute rejection in renal transplant recipients – development and optimization of a urinary proteomic approach" ?

Further prospective studies are therefore needed in larger populations, where biopsies also are performed in the control patients, in order to elucidate on the involvement of these proteins in acute rejection and their potential usability as diagnostic biomarkers. The use of urine and a trend towards an increase of proteins levels prior to deterioration of graft function potentially opens for early, specific and non-invasive detection of acute rejection episodes.