• No results found

Development of an analysis method for a glycosylated protein using MALDI-MS and separation techniques

N/A
N/A
Protected

Academic year: 2021

Share "Development of an analysis method for a glycosylated protein using MALDI-MS and separation techniques"

Copied!
62
0
0

Loading.... (view fulltext now)

Full text

(1)

DEGREE PROJECT IN CHEMICAL ENGINEERING, FIRST CYCLE, 15 HP

STOCKHOLM, SWEDEN 2020

Development of an analysis method for a glycosylated protein using MALDI-MS and separation techniques

JESSICA SINGH

(2)

DEGREE PROJECT

Bachelor of Science in Chemical Engineering

Title: Development of an analysis method for a glycosylated protein using MALDI-MS and separation techniques.

Swedish title: Utveckling av en analysmetod för ett glykosylerat protein med MALDI-MS och separationstekniker

Keyword: Immunoglobulin G, glycopeptide enrichment, Glycans, MALDI, CE-MS

Work place: Royal Institute of technology, KTH

Supervisor at

work place: Yuye Zhou

Supervisor at

KTH: Åsa Emmer

Student: Jessica Singh

(3)

Abstract

The antibody Immunoglobulin G (IgG) main function is to protect and prevent the body from infections, and it is normally found in human serum. This study is about IgG glycosylation, which is associated with different types of diseases such as neurological diseases, cancers and immunodeficiency etc. This study attempts to optimize IgG glycopeptide enrichment in a 100 µL micropipette tip set up, and to separate the enriched glycopeptides using capillary electrophoresis (CE). Matrix-assisted laser desorption/ionization – mass spectrometry (MALDI-MS) was used for data acquisition and glycopeptide profiling.

In this study, loading solutions with different combinations of acetonitrile (ACN) and trifluoroacetic acid (TFA), together with various precondition and sample preparation procedures were evaluated on IgG digest samples. Best enrichment performance, particularly regarding the selectivity, was achieved using the parameters as follows: loading solution of 83% ACN/16%

H2O/1% TFA, sample solution in H2O containing 83% ACN, using a 100 µL micropipette tip packed with 1 mg cotton wool. A re-enrichment step was carried out on enriched glycopeptide samples, and improved selectivity of glycopeptides could be observed. Enriched glycopeptides could be separated into three major groups by CE using an acidic background electrolyte of 50 mM formic acid and 50 mM acetic acid, pH 2.5.

(4)

Sammanfattning

Huvudfunktionen för antikroppen Immunoglobulin G (IgG) är att skydda kroppen och förhindra infektioner och det finns normalt i mänskligt serum. Denna studie handlar om glykosylering, som är kopplad till olika typer av sjukdomar såsom neurologiska sjukdomar, cancer och immunbrist etc, och är en potentiell biomarkör för sjukdomar. Denna studie försöker optimera en IgG glycopeptidanrikningsmetod i en 100 µL mikropipettspets och separera de anrikade och intakta glycopeptiderna med hjälp av kapillärelektrofores (CE). Matrisassisterad laserdesorption/jonisering – masspektrometri (MALDI-MS) användes för datainsamling och glykopeptidprofilering

I denna studie utvärderades lösningar med olika kombinationer av acetonitril (ACN) och trifluororättiksyra (TFA) tillsammans med olika föberedelseförfaranden och provberedningsprocedurer på IgG prover. Anrikningsprestanda, särskilt selektiviteten, uppnåddes bäst med användning av följande parametrar: lösningen av 83% ACN / 16% H2O / 1% TFA, provlösning i H2O innehållande 83% ACN, med användning av en 100 mikroliter mikropipettspets fylld med 1 mg bomull. Återreningssteget genomfördes på anrikade glykopeptidprover och förbättrad selektivitet för glykopeptider kunde observeras. Anrikade glykopeptider kunde separeras i tre huvudgrupper med CE med användning av sur bakgrundselektrolyt med 50 mM myrsyra och 50 mM ättiksyra, pH 2,5.

(5)

Acknowledgements

It is with great pride I hereby present my bachelor thesis. I have gained a lot of knowledge of this research and which has been beneficial to my development in my career and my studies in the future. I would like to express my most sincere gratitude and appreciate to my supervisor, Yuye Zhou, a great mentor, for her direction, inspiration and persistent support in my undertaking of this research.

My sincere gratitude also goes to my examiner, Professor Åsa Emmer, for giving me the opportunity to be part of this research and for her constant kindness and support. I also want to express my appreciation of the PhD- students Joakim Romson and Linus Svenberg and master student Leo Sjöström and Dr. LeilaJosefsson who all put in the effort for making me feel welcomed in the department.

Finally, I want to express my heartfelt gratitude to my parents and my brother, Dennis, for the moral support and love.

(6)

Table of Contents

1 Introduction ... 1

2 Theoretical Background ... 2

2.1 Immunoglobulin G (IgG) ... 2

2.2 MALDI – MS ... 3

2.3 CE ... 5

3 Experimental Methods ... 6

3.1 Chemical, solvent and instrument information ... 6

3.2 IgG Glycopepetide enrichment ... 6

4 Results and discussion ... 8

4.1 Selection of best parameters for enriched glycopeptide identification ... 8

4.1.1 IgG glycopeptide separation using CE ... 28

5 Conclusions ... 29

6 References ... 30

7 Appendix ... 32

7.1 Appendix A ... 32

(7)

1 Introduction

The antibody Immunoglobulin G (IgG) main function is to protect and prevent the body from infections, and it is found in human serum. IgG molecules are created and then released by plasma B cells. Every IgG molecule has two antigen binding sites. This project was about the study of IgG glycosylation. IgG glycosylation is related to different diseases such as neurological diseases, cancers and immunodeficient etc, and is a potential disease biomarker. Glycosylation is an important mechanism of secondary protein processing within cells and it is a central part in being able to determine protein structure, function and stability. Glycosylation affects the three- dimensional configuration of proteins, which is important when there is a protein-protein interaction occurring, such as for protein ligands and their corresponding receptors or when creating large macromolecules. [1]

The aim of this project was to optimize an IgG glycopeptide enrichment protocol using cotton wool tips to separate the abundant non-glycopeptides from intact glycopeptides. MALDI-MS was used for data acquisition and glycopeptide identification of the enriched glycopeptides. CE, which was used in this project, is a separation technique used for separation of glycopeptides according to their sizes and charges. CE is used when the detection and identification of individual analytes in a sample mixture is difficult and separation is needed. Compared to liquid chromatography, also a commonly used separation technique, CE requires only µL sized sample volume. It is thus preferred in miniaturized biological sample analysis. [2]

(8)

2 Theoretical Background

2.1 Immunoglobulin G (IgG)

Immunoglobulins (Igs), are plasma proteins that are normally created in B-cells. [2] The Igs are arranged into various isotypes relying upon their capacities classified into different isotypes depending on their functions like IgA, IgD, IgE, IgG and IgM. The Ig which is concentrated on in this report is IgG. IgG is a sort of antibody of which the primary function is to shield and keep the body from various diseases, and it is mostly found in human serum. There are subclasses of IgG and they are IgG1, IgG2, IgG3 and IgG4. [4]

IgG recognize and kills pathogens which sometimes help out with other molecules. When the body recognizes an unfamiliar molecule, antigen bind to an antigen-binding site and attached to a pathogen. [5]

IgG is made up of four peptide chains, two heavy peptides (50 kDa), which are connected together and to a light chain by disulphide bonds, and two light peptide (25 kDa) chains. The Y-shape is created due to the tetramer has two identical parts [2] Each arm of the Y-shaped IgG has two identical parts containing an antigen binding site.

They are called Fragment antigen binding, Fab, if cleaved off. The body of the Y is called fragment crystallizable, Fc, due to its crystallizable characteristics. [6] This can be found in the fundamental structure of IgG in Figure 1. The IgG structure has an intersection, which is known as the

hinge region. The basic contrast between the IgG subclasses are the measure of disulphide bridges in the hinge region. There are 2,4,11 and 4 disulphide bridges for IgG1, IgG2, IgG3 and IgG4 individually due to the different number of amino residues. [3]

In this study, prior to analysis, IgG was cleaved into smaller peptides with tryptic digestion at 37

°C for 17h. This was performed by the PhD-student Yuye Zhou.

Figure 1. Structure of Immunoglobulin G [7]

(9)

2.2 MALDI – TOF-MS

MALDI is an ionization method, which utilizes a matrix that is energy absorbing to be able to create ions from larger molecules with minimal fragmentation. [9]

Direct ionizing molecules caused destruction or extensive fragmentation of analytes as the added energy usually was excess, hence MALDI was created to prevent his. Nonetheless, in MALDI, a matrix is available along with the analyte and absorbs most of the energy provided by the UV-laser. The laser excites the matrix molecules and makes a plume of the matrix and sample to evaporate from the surface, see figure 2. The mechanism of protonation of analytes are not completely understood however this is the step where the analytes become ionized. The laser adds energy quickly (less

likely to corrupt the analytes if the process is quick) to the sample and raises the temperature to 1000 C°, which

causes explosive evaporation. [10]

The matrix is generally made up of small, aromatic

molecules with either acidic or basic functional groups relying on the sample and analyte qualities.

Both the analyte and the matrix solutions are deposited onto a target plate made of steel, where several of sample spots can be

deposited for analysis. The solvent on the target plate evaporates, which cause formation of crystals and the analytes are embedded and co- crystallized. [12]

The results given by the MALDI-TOF (Time of flight) separation are

Figure 2. How MALDI works [11]

(10)

off chance that all the ions start their path simultaneously or within a brief timeframe, the lighter ions show up sooner than the heavier ions to the detector. This translates into the mass spectrum.

[13] A mass spectrum is a graph, which incorporates the distribution and intensity of the ions introduced to the mass analyser, separated according to the m/z values. The x-axis reflects the m/z and y-axis reflects the intensity (abundance). [12] In figure 3, the basic features of a mass spectrum are shown.

(11)

2.3 CE

CE could be viewed as a specific sort of liquid separation technique. The CE system contains a high voltage supply with electrodes that are lowered into vials with background electrolyte (BGE), a fused silica capillary, and a detector (the sample and the setup is seen in figure 4). The sample is either electrokinetically or hydrodynamically infused into the capillary. The separation of the analytes occurs inside the capillary, and the analytes are detected while passing the detector. The detector most commonly used

is the UV detector, which estimates absorbance of the analyte as it passes the detection window. [10]

In CE, ions migrate in the solution through a fused silica capillary via an electric field of high potential (kV). Thereafter, the charged species migrates in various velocities in the solution, where the result is represented in a plug-like component profile due to the electro-osmotic flow, which is created by the electric field. [12]

The BGE is significant in CE as its function is to give consistent conditions for the analyte ions during separation, and to make these conditions independent from the sample composition. [2] The most regularly utilized method of CE is capillary zone electrophoresis (CZE), where the BGE is frequently a solution of a buffer salt, which can contain e.g. organic solvents. [10]

In CE, the capillaries utilized are made of fused silica, covered on the outside with a thin layer of polyimide, which improves the adaptability and lessen the fragility of the glass. The length and inner diameter can differ depending on what fits the experiment. [10]

Figure 4 Model of setup for CE-MS experiments

(12)

3 Experimental Methods and Material

3.1 Chemical, solvent and instrument information

ACN, TFA, IgG (IgG from human serum), Eppendorf LoBind® microcentrifuge tubes (Lobind tube) were purchased from Sigma-Aldrich (Stockholm, Sweden). Water (MQ H2O) was purified to a resistivity of 18.2 MΩ·cm (25 °C) in a Millipore Synergy® 185 (Bedford, MA, USA).

Pierce® C18 Tips, 100 µL bed size were from purchased from Thermo Fisher Scientific (Rockford, USA). The ultrafleXtreme MALDI-TOF/TOF, the MALDI matrix 2,5-

dihydroxybenzoic acid (DHB) and MALDI plates ground steel (GS) were purchased from Bruker Daltonics (Bremen, Germany).

3.2 IgG Glycopeptide enrichment Cotton tips preparation

2 mg cotton obtained from a cotton swab was pushed into the tip of a 100 µL pipette. The cotton should not be too tight in the tip as it will make it difficult to aspirate any liquid trough the tip.

Alternatively, the end of the tip of the pipetted can be cut wider.

Glycopeptide enrichment

A cotton tip was preconditioned with 100 µL loading solution five times. Evaluated sample solutions and loading solutions were listed in the results and discussion section for each experiment. Thereafter, the sample solution was pipetted up and down 20 times through the cotton tip to allow glycoconjugate adsorption. The cotton tip was washed several times with loading solution. Lastly, the analytes adsorbed in the cotton tip was eluted into a new vial with 100 µL H2O, and this was saved as elution fraction. The elution fraction was dried in na Eppendorf ® Centrifuge and reconstituted with 5 µL H2O. All samples were stored in freezer until further analysis on MALDI-MS.

Glycopeptide profiling using MALDI-MS

0.5 µL of sample was spotted on a target plate. Then, 0.5 µL DHB matrix (20 mg/mL in in ACN/TFA) was applied after the sample spot was dried. Ultraflex MALDI-TOF-MS, which is equipped with a Smartbream-II-laser (355 nm,UV) was used for this experiment. The mode used for analyzing was positive reflectron mode. The laser intensity was at 60%. For each sample, 3

(13)

Glycopeptide separation using CE

A 60 cm capillary was prepared with an effective length of 41.5cm. The detection window was made by burning and removing the polymer coating on the outside of the capillary. 50 mM formic acid and 50 mM acetic acid (pH 2.5) was used as BGE. Before run, the capillary was flushed with 1 M NaOH for 30 min, H2O for 30 min, and BGE for 30 min. Enriched IgG glycopeptide sample was injected by pressure at 100 mbar for 5s. The separation was carried out at 20 KV.

(14)

4 Results and discussion

4.1 Selection of the best parameters for enriched glycopeptide identification Experiment 1

Evaluated loading solutions and sample mixtures used in experiment 1 are listed in Table 1. In order to evaluate the selectivity, elution fractions from all samples were accessed to see if non- glycopeptides could be separated from glycopeptides.

Table 1. Experiment 1 – sample and loading solutions tested. All IgG digest used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading Solution

1 86 µL of ACN, 8 µL H2O, 1

µL TFA and 5 µL IgG digest

86% ACN/13% H2O/1%

TFA

2 86 µL of ACN, 8 µL H2O, 1

µL 10% TFA and 5 µL IgG digest

86% ACN/13.9% H2O/0.1%

TFA

3 89 µL of ACN, 5 µL H2O, 1

µL TFA and 5 µL IgG digest

89% ACN/10% H2O/1%

TFA

4 89 µL of ACN, 5 µL H2O, 1

µL 10% TFA and 5 µL IgG digest

89% ACN/10.9% H2O/0.1%

TFA

5 83 µL of ACN,11 µL H2O, 1

µL TFA and 5 µL Sample IgG digest

83% ACN/16% H2O/1%

TFA

6 83 µL of ACN, 11 µL H2O, 1

µL 10% TFA and 5 µL Sample IgG digest

83% ACN/16.9% H2O/0.1%

TFA

(15)

a) b)

c) d)

e) f)

Figure 5 MALDI-MS spectra of elution fraction, glycopeptides mainly located in the range of m/z 2600-3000,from sample 1-6: a) sample 1, b) sample 2, c) sample 3, d) sample 4, e) sample 5 and f) sample 6.

It can be seen from Figure 5b), 5d) and 5f) that samples containing 0.1% TFA have a lower intensity of glycopeptides compared to sample 5a), 5c) and 5f) containing 1% TFA.

Spectra of elution fractions from samples 3 and 4 (Figures 5c and d) show a lot of contamination from non-glycopeptides (between m/z range 1000-1500 and 2100-2500) when sample and loading solutions contained 89% ACN. For further optimization, more experiments on sample and loading solutions containing 86% and 83% ACN with 1% TFA were carried out.

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(16)

Experiment 2

In experiment 2, loading solutions containing 86% and 83% ACN were compared. The amount of IgG digest in the samples was also investigated. All sample and loading solution information are listed in Table 2. In this experiment, in order to obtain better results, washing repetition was increased to 10 times from previously 5 times in experiment 1.

Table 2. Experiment 2 – sample and loading solutions tested. All IgG digest samples used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution

1 and 2 83 µL of ACN, 8.5 µL H2O,

1 µL TFA and 7.5 µL IgG digest

83% ACN/16% H2O/1%

TFA

3 and 4 83 µL of ACN, 6 µL H2O, 1

µL TFA and 10 µL IgG digest

83% ACN/16% H2O/1%

TFA

5 and 6 86 µL of ACN,5.5 µL H2O, 1

µL TFA and 7.5 µL Sample IgG digest

86% ACN/13% H2O/1%

TFA

7 and 8 86 µL of ACN, 3 µL H2O, 1

µL TFA and 10 µL Sample IgG digest

86% ACN/13% H2O/1%

TFA

a) b)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0 1 2 3 4 5 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(17)

c) d)

Figure 6 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from sample 1-8 a) sample 1, b) sample 3, c) sample 6, and d) sample 8.

In figure 6a and 6b, a lot of contamination from non-glycopeptides were detected when the sample and loading solutions contained 83% ACN, and 7.5 µg IgG digest was loaded, due to increased amount of IgG digest compared to previous experiments. The results were unrepeatable between sample 6c and 6d, probably due to bad cotton wool tip preparation. Bad cotton wool tip preparation could be caused by packing the cotton wool in the 100 µl tip too tight or too loose making the glycopeptides not get absorbed by the cotton wool or making it difficult to be able to aspirate any liquid trough the tip. Therefore, the comparison between different loading solutions and loading amount of IgG digest was further carried out in following experiments. In 6d, the intensity is higher as there is more amount of IgG digest added to the sample compared to 6c) where less IgG digest is added. In 6d, there is less contamination of non-glycopeptides compared to 6c, b and a. From what can be observed in the spectra in Figure 6, the more amount of IgG digest, and a sample and loading solution containing 86% ACN, will have a higher intensity of glycopeptides in the range of m/z 2600-3000 and a lower amount of contaminations of non-glycopeptides in the elution fraction.

0 2 4 6 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.00 0.25 0.50 0.75 1.00 1.25 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(18)

Experiment 3

In experiment 3, the comparison was carried out between 7.5 and 5 µL loaded IgG digest, and between 1% and 3% TFA in the loading solution. All the sample and loading information are listed in table 3.

Table 3. Experiment 3 – sample and loading solution parameters tested. All IgG digest used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution

1 and 2 83 µL of ACN, 11 µL H2O, 1

µL TFA and 5 µL IgG digest

83% ACN/16% H2O/1%

TFA

3 and 4 83 µL of ACN, 8.5 µL H2O,

1 µL TFA and 7.5 µL IgG digest

83% ACN/16% H2O/1%

TFA

5 and 6 83 µL of ACN, 9 µL H2O, 3

µL TFA and 5 µL Sample IgG digest

83% ACN/14% H2O/3%

TFA

7 and 8 83 µL of ACN,6.5 µL H2O, 3

µL TFA and 7.5 µL Sample IgG digest

83% ACN/14% H2O/3%

TFA

(19)

a) b)

c) d)

Figure 7 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from sample 1-8: a) sample 1, b) sample 4, c) sample 6, and d) sample 8.

It could be observed that sample 7a and 7b, which contain the loading solution with 1% TFA, have a higher intensity of glycopeptides compared to 6d. It could also be observed from Figure 7 that a lot of non-glycopeptides were eluted out together with glycopeptides when the loading solution contained 3% TFA and can especially be seen in 7c. In order to obtain better selectivity, 1% TFA in the loading solution is thus preferred. When using a higher amount of IgG digest, there are more non-glycopeptide eluted together with glycopeptides, which can be observed in 6b and 6c) but the intensity is higher. The reasoning for higher amount of non-glycopeptides present could be due to factors such as bad cotton wool tip preparation.

0 1 2 3 4 5 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0 1 2 3 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.5 1.0 1.5 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.5 1.0 1.5 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(20)

Experiment 4

In experiment 4, a loading solution with 80% ACN was compared in combination with different proportions of TFA, 0.1% or 1% . All the sample and loading solution information is listed in table 4.

Table 4. Experiment 4 – sample and loading solution parameters tested. All IgG digest used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution

1 and 2 80 µL of ACN, 11.5 µL H2O,

1 µL 10% TFA and 7.5 µL IgG digest

80% ACN/19.9% H2O/0.1%

TFA

3 and 4 80 µL of ACN, 11.5 µL H2O,

1 µL TFA and 7.5 µL IgG digest

80% ACN/19% H2O/1%

TFA

a) b)

Figure 8 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from sample 1-4: a) sample 1 and b) sample 4

It could be seen from Figure 8 that most of the non-glycopeptides were excluded from the glycopeptides. These loading solutions were further evaluated and compared with loading solutions containing 83 % ACN.

0 1 2 3 4 5 6 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.5 1.0 1.5 2.0 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(21)

Experiment 5

In experiment 5, the samples from previous experiments, which gave the best results, were compared to each other. The comparison between 80% ACN and 83% ACN, between 0.1% and 1% TFA were performed (Table 5).

Table 5. Experiment 5 – sample and loading solution parameters tested. All IgG digest used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution

1 and 2 80 µL of ACN, 11.5 µL H2O,

1 µL TFA and 7.5 µL IgG digest

80% ACN/19% H2O/1%

TFA

3 and 4 80 µL of ACN, 11.5 µL H2O,

1 µL 10% TFA and 7.5 µL IgG digest

80% ACN/19.9% H2O/0.1%

TFA

5 and 6 83 µL of ACN, 8.5 µL H2O,

1 µL TFA and 7.5 µL IgG digest

83% ACN/16% H2O/1%

TFA %

7 and 8 83 µL of ACN, 8.5 µL H2O,

1 µL 10% TFA and 7.5 µL IgG digest

83% ACN/16.9% H2O/0.1%

TFA

(22)

a) b)

c) d)

Figure 9 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000, from sample 1-8: a) sample 2, b) sample 3 c) sample 6 and d) sample 8

In Figures 9a and 9b, samples containing 1% TFA had a higher intensity of enriched

glycopeptides, compared to samples containing 0.1% TFA. This could also be observed from Figures 9c and 9d, where higher glycopeptide intensities were obtained when 1% TFA and 83%

ACN were used in sample and loading solutions, compared to that of 0.1% TFA and 83% ACN.

It can be concluded from Figure 9 that 1% TFA gave a better result compared to 0.1% TFA.

0.0 0.5 1.0 1.5 2.0 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.5 1.0 1.5 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 1.0 5 x10

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0 2 4 6 8 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(23)

Experiment 6

In experiment 6, the samples from previous experiments, which gave the best results were compared, and an additional wetting step (5 times with 100 µL H2O) before pre-conditioning was also investigated to see if better results could be obtained. Information about sample and loading solutions, and additional information about the steps are listed in Table 6.

Table 6. Experiment 6 – sample and loading solution parameters tested. All IgG digest used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution Additional

information 1 and 2 80 µL of ACN, 11.5

µL H2O, 1 µL TFA and 7.5 µL IgG digest

80% ACN/19%

H2O/1% TFA

3 and 4 80 µL of ACN, 11.5 µL H2O, 1 µL TFA and 7.5 µL IgG digest

80% ACN/19%

H2O/1% TFA

Additional wetting step

5 and 6 83 µL of ACN, 8.5 µL

H2O, 1 µL TFA and 7.5 µL IgG digest

83% ACN/16%

H2O/1% TFA

7 and 8 83 µL of ACN, 8.5 µL

H2O, 1 µL TFA and 7.5 µL IgG digest

83% ACN/16%

H2O/1% TFA

Additional wetting step

(24)

a) b)

c) d)

Figure 10 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from: a) sample 2, b) sample 3 c) sample 6 and d) sample 8

From the spectra it could be seen that Figure 10b shows the best results, where 80% ACN and the additional wetting step was applied. Figure 10a shows a larger amount of contamination from non-glycopeptides. In Figures 10c and 10d, 83% ACN was used and the intensity of

glycopeptides was lower compared to when 80% ACN was used, seen in figure 10b. The

additional wetting step worked less efficiently when using 83% ACN. The additional wetting step seems to give a slightly better result but the 83% ACN gave less non-glycopeptides. More data has to be gathered to be able to confirm this.

0.0 0.5 1.0 1.5 2.0 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.5 1.0 1.5 2.0 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.5 1.0 1.5 2.0 2.5 3.0 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.5 1.0 1.5 2.0 2.5 3.0 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(25)

Experiment 7

In experiment 7, trials were carried out with the same loading solution (83% ACN/16% H2O/1%

TFA) but different sample solutions. It aimed to investigate if it is necessary to have 1% TFA in sample solution. Information about sample and loading solutions, and additional information of the steps are listed in Table 7.

Table 7. Experiment 7 – sample and loading solution parameters tested. All IgG digest used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution Additional

information

1 and 2 83 µL of ACN, 8.5 µL

H2O, 1 µL TFA and 7.5 µL IgG digest

83% ACN/16%

H2O/1% TFA

3 and 4 83 µL of ACN, 8.5 µL

H2O, 1 µL TFA and 7.5 µL IgG digest

83% ACN/16%

H2O/1% TFA

Additional wetting step

5 and 6 83 µL of ACN, 9.5 µL

H2O and 7.5 µL IgG digest

83% ACN/16%

H2O/1% TFA

7 and 8 83 µL of ACN, 9.5 µL

H2O and 7.5 µL IgG digest

83% ACN/16%

H2O/1% TFA

Additional wetting step

(26)

a) b)

c) d)

Figure 11 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from: a) sample 2, b) sample 3 c) sample 6 and d) sample 7

It could be seen from Figure 11 that the additional wetting step resulted in lower selectivity, with more non-glycopeptides detected in the elution fraction (Figures 11b and 11d). It could also be seen that the spectra from samples without TFA gave better results (Figures 11c and 11d).

Therefore, to obtain the best results, the loading solution 83% ACN/16% H2O/1% TFA, and sample solution containing 83% ACN without TFA are recommended and used for following tests.

0.0 0.2 0.4 0.6 0.8 1.0 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 1.0 5 x10

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.00 0.25 0.50 0.75 1.00 1.25 1.50 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.00 0.25 0.50 0.75 1.00 1.25 1.50 5 x10

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(27)

Experiment 8

12 samples prepared and analyzed using the recommended parameters. Repeatable results could be obtained and are shown in Figure 12.

a) b)

c) d)

Figure 12 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from: a) sample 5, b) sample 6, c) sample 11 and d) sample 12

In Figure 12, some non-glycopeptides in the low mass range could still be observed, hence repeated enrichment on enriched glycopeptide samples was carried out in order to get as pure glycopeptides as possible.

Enriched glycopeptides from sample 1 and sample 2 were combined to a total volume of 10 µL, and thereafter 83 µL ACN and 7 µL H2O were added. This was also done for the rest of the samples, and in the end there were 6 samples. Then the glycopeptide enrichment steps were applied again on enriched samples with 5 washing repetitions. After re-enrichment, purer spectra with less non- glycopeptides could be obtained as shown in Figure 13.

0.0 0.5 1.0 1.5 2.0 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0 2 4 6 8 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0 1 2 3 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.5 1.0 1.5 2.0 2.5 4 x10

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(28)

Figure 13 MALDI-MS spectrum of the elution fraction from a sample after re-enrichment, glycopeptides mainly located in the range of m/z 2600-3000,

The re-purification in figure 13 gave slightly better results compared to the spectra in figure 12 with similar intensity of glycopeptides and a lower intensity of non-glycopeptides that occur between the range of m/z 0-1500. This step is a not as significant part of the procedure since the results are quite the similar without the purification. Hence, this step can be used if needed, especially if there is a lot of unmodified peptides present.

0.0 0.5 1.0 1.5 2.0 2.5 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(29)

Experiment 9

In experiment 9, the wash procedure during glycopeptide enrichment was changed to see if the purification of glycopeptides would be better compared to the previous method. The results shown are from the spot which gave the best results, and the results of the worse replica will not be shown.

Information about sample and loading solutions, and additional information are listed in Table 8.

Table 8. Experiment 8 – sample and loading solution parameters tested. All IgG digests used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution Washing repetition

1 83 µL of ACN, 7 µL

H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

10

2 83 µL of ACN, 7 µL

H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

10

3 83 µL of ACN, 7 µL

H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

10

4 83 µL of ACN, 7 µL

H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

10

5 83 µL of ACN, 7 µL

H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

10

6 83 µL of ACN, 7 µL

H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

15

7 83 µL of ACN, 7 µL 83% ACN/16% 15

(30)

9 83 µL of ACN, 7 µL H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

15

10 83 µL of ACN, 7 µL

H2O and 10 µL IgG digest

83% ACN/16%

H2O/1% TFA

15

During the washing step, aspiration and dispense 15 times of the loading solution was performed instead of 10 times. The rest of the procedure is the same as in the previous experiments.

a) b)

Figure 14 MALDI-MS spectra of elution fraction majorly located in the range of m/z 2600-3000from sample 1-10: a) washing repetition, 10 times and b) washing repetition, 15 times

What can be seen in the spectra in Figure 14 is that there is no obvious difference in intensities.

The intensities of the glycopeptides are equal in both 14a) and 14b). When changing the method with adding more washing repetitions, slightly less non-glycopeptides appears. This step is not necessary and does not benefit the analysis method and can be disregarded.

0.0 0.5 1.0 1.5 2.0 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.5 1.0 1.5 2.0 2.5 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(31)

Experiment 10

In experiment 10, 10 µL tips packed with 0.5 mg cotton wool were utilized for investigation of the possibility to improve the result if a small sample volume was applied. The loading solution and sample solution information are listed in Table 9.

Table 9. Experiment 10 – sample and loading solution parameters tested. All IgG digest used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution

1 8.3 µL of ACN, 0.7

µL H2O and 1 µL IgG digest

83% ACN/16%

H2O/1% TFA

a) b)

Figure 15 MALDI-MS spectra of non-binding and elution fraction from sample 1:a) non-binding fraction b) elution fraction

The parameters optimized with 100 µL tips could also be used for 10 µL tips, which is seen in figure 15b. In previous experiments in this report 100 µL tips were used to be able to create the sample and loading solution and preform the glycopeptide enrichment. This can also be obtained using 10 µL tips. The spectra in 15b has similar intensity of glycopeptides to 14a in figure 14..

With the 10 µL tips non-glycopeptides could also be eliminated and glycopeptides could be enriched. The benefits to obtaining the same results with different size of the tips is that less product is used causing less waste, meaning it is more sustainable.

0.00 0.25 0.50 0.75 1.00 1.25 1.50 5 x10

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.00 0.25 0.50 0.75 1.00 1.25 5 x10

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(32)

Experiment 11

In experiment 11, the same approach which was applied for experiment 13 to obtain better results was used for the sample with 80% and 86% ACN. The loading solution and the sample solution information are listed in Table 10.

Table 10. Experiment 11 – sample and loading solution parameters tested. All IgG digests used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution

1,2,3,4 and 5 8.6 µL of ACN, 0.4 µL H2O and 1 µL IgG digest

86% ACN/13% H2O/1%

TFA 6,7,8,9 and 10 8.0 µL of ACN, 1 µL H2O

and 1 µL IgG digest

80% ACN/19% H2O/1%

TFA

a) b)

c)

Figure 16 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from: a) sample with 83% ACN b) sample with 86% ACN and c) sample with 80% ACN.

The results turned out better when the approach was changed. What could be seen in figure 16 b) and c) is that the sample with 86% ACN has more contaminations of non-glycopeptides in the elution fraction compared to the sample with 80% ACN. Both samples have higher intensities of

0.00 0.25 0.50 0.75 1.00 1.25 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 1.0 1.2 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0 1 2 3 4 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(33)

Experiment 12

In experiment 15, a 100 µL tip packed with 1 mg cotton wool was evaluated and compared with the results from a 2 mg cotton wool tip. The loading solution and sample solution information are listed in Table 11.

Table 11. Experiment 12 – sample and loading solution parameters tested. All IgG digests used had a concentration corresponding to 1 mg/mL IgG.

Sample nr. Sample Loading solution

1 83 µL of ACN, 7 µL H2O and

10 µL IgG digest

83% ACN/16% H2O/1%

TFA

a)

b)

Figure 17 MALDI-MS spectra of elution fractions, glycopeptides mainly located in the range of m/z 2600-3000,from a)

0.0 0.5 1.0 1.5 2.0 x104

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.5 1.0 1.5 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(34)

4.1.1 IgG glycopeptides separation using CE Run 1

Figure 18 CE electropherogram of enriched IgG glycopeptides. Injection: 100 mbar 5s, Separation voltage: 20 KV, BGE: 50 mM formic acid, 50 mM acetic acid, pH 2.5.

a) b)

c)

Figure 19 The three different structure glycoforms in order a) G0F b) G1F c) G2F

In the CE electropherogram, figure 18, some separated peaks can be detected around 19 minutes.

The intensity of the peaks range between 0.8 mAu and >1 mAu. The separation result was compared with results found in the literature. [15] In the literature, there are three main peaks in a CE electropherogram of IgG glycopeptides after separation. They are defined as three different glycoforms, G0F, G1F and G2F, seen in figure 19, corresponding to m/z of respectively 878.7,

(35)

This shows that enriched glycopeptides can be separated into three groups by CE, which agreed with results from the literature.

5 Conclusion

In this study, experiments were performed to find the best parameters for IgG glycopeptide enrichment using 100 µL micropipette tips packed with cotton. The best result was obtained when using a loading solution of 83% ACN/16% H2O/1% TFA and a sample solution prepared with 83%

ACN, without TFA using x mg of cotton. These parameters gave the highest intensity of enriched glycopeptides, and the lowest amount of non-glycopeptides. The re-purification step can be used if needed e.g. to purify a sample, which have high amounts of non-glycopeptides. In this study, CE was used to separate the glycopeptides, and what could be seen was that enriched glycopeptides can successfully be separated into three major groups according to different glycosylation patterns in agreement with the literature.

(36)

6 References

[1] Brian J. Arey (2012). The Role of Glycosylation in Receptor Signaling, Glycosylation, Stefana

Petrescu, IntechOpen, DOI: 10.5772/50262. Available from:

https://www.intechopen.com/books/glycosylation/the-role-of-glycosylation-in-receptor-signaling [2] Gimenez. E, Ramos-Hernan.R, Benavente. F, Barbosa. J, Sans-Nebot. V (2012) “Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray–time of flight-mass spectrometry”. Analytica Chimica Acta. Elsevier. DOI:

https://doi.org/10.1016/j.aca.2011.10.028

[3] Janeway, C. A., Travers, P., Walport, M., Walport, M., Capra, J., D. (1999) Immuno biology the immune system in health and disease, 4th ed., London: Elsevier Science/Garland, ISBN: 0- 8154-32173, p.79-81

[4] Vidarsson, G., Dekkers, G., Rispens, T. (2014) IgG subclasses and allotypes: from structure and functions, Frontiers in immunology 5:1-17

[5] Jefferis, R. (1990) Molecular structure of human IgG subclasses. Nottingham: Elsevier. ISBN:

9780-08-037504-5

[6] Janeway, C. A., Travers, P., Walport, M., Walport, M., Capra, J., D. (2001) Immuno biology the immune system in health and disease, 5th ed., New York: Elsevier Science/Garland, ISBN:978081533-642-6, ch. 3

[7] Commons.wikimedia.org. 2013. File:2220 Four Chain Structure Of A Generic Antibody-Igg2 Structures.Jpg - Wikimedia Commons. [online] Available at:

<https://commons.wikimedia.org/wiki/File:2220_Four_Chain_Structure_of_a_Generic_Antibody -IgG2_Structures.jpg> [Accessed 15 September 2020].

[8] Alagesan, K., Khilji, S. K., & Kolarich, D. (2017). It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment. Analytical and bioanalytical chemistry, 409(2), 529–538. https://doi.org/10.1007/s00216-016-0051-6

[9] Hillenkamp, F; Karas, M; Beavis, R C.; Chait, B T. (1991). "Matrix-assisted laser desorption/ionization mass spectrometry of biopolymers". Analytical Chemistry. 63 (24): 1193A–

1203A. doi:10.1021/ac00024a002. ISSN 0003-2700. PMID 1789447

(37)

[12] Greaves, J., Roboz, J. (2014). Mass spectrometry for the novice. Boca Raton, CRC Press, ISBN: 978-1-4200-94183, p.67-70, 6-12

[13] Proteomics, C., n.d. MALDI-TOF Mass Spectrometry. [online] Creative Proteomics. Available at: <https://www.creative-proteomics.com/technology/maldi-tof-mass-spectrometry.htm>

[Accessed 10 May 2020].

[14] Kok.W. (2000) The Background Electrolyte. In: Capillary Electrophoresis: Instrumentation and Operation. Chromatographia CE-Series, vol 4. Vieweg+ Teubner Verlag, Wiesbaden. DOI:

https://doi.org/10.1007/978-3-322-83133-0_7 ISBN: 978-3-322-83133-0

[15] Kammeijer GS, Kohler I, Jansen BC, et al. Dopant Enriched Nitrogen Gas Combined with Sheathless Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry for Improved Sensitivity and Repeatability in Glycopeptide Analysis. Anal Chem. 2016;88(11):5849‐5856.

doi:10.1021/acs.analchem.6b00479

(38)

7 Appendix

7.1 Appendix A

Experiment 1

a)

b)

1172.818

1493.855

2602.417 825.664

2958.529 923.065

2926.534

2163.383 2441.410

3389.871

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

(39)

c)

d)

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.6 0.8 x105

Intens. [a.u.]

0.6 0.8 x105

Intens. [a.u.]

(40)

e)

f)

Figure 22 MALDI-MS spectra of elution fractions from sample 1-6: a) sample 1 (spot 1, spot 2 and spot 3), b) sample 2(spot 1, spot 2 and spot 3), c) sample 3(spot 1, spot 2 and spot 3), d) sample 4(spot 1, spot 2 and spot 3), e) sample 5 (spot 1, spot 2 and spot 3) and f) sample 6(spot 1, spot 2 and spot 3)

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z 0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

0.0 0.2 0.4 0.6 0.8 x105

Intens. [a.u.]

1000 1500 2000 2500 3000 3500 4000 4500

m /z

References

Related documents

46 Konkreta exempel skulle kunna vara främjandeinsatser för affärsänglar/affärsängelnätverk, skapa arenor där aktörer från utbuds- och efterfrågesidan kan mötas eller

Inom ramen för uppdraget att utforma ett utvärderingsupplägg har Tillväxtanalys också gett HUI Research i uppdrag att genomföra en kartläggning av vilka

Tillväxtanalys har haft i uppdrag av rege- ringen att under år 2013 göra en fortsatt och fördjupad analys av följande index: Ekono- miskt frihetsindex (EFW), som

Som rapporten visar kräver detta en kontinuerlig diskussion och analys av den innovationspolitiska helhetens utformning – ett arbete som Tillväxtanalys på olika

I regleringsbrevet för 2014 uppdrog Regeringen åt Tillväxtanalys att ”föreslå mätmetoder och indikatorer som kan användas vid utvärdering av de samhällsekonomiska effekterna av

Det finns en risk att samhället i sin strävan efter kostnadseffektivitet i och med kortsiktiga utsläppsmål ’går vilse’ när det kommer till den mera svåra, men lika

The co-feature ratio approach has been successfully used for evaluating sample preparation methods [III] and for comparing chromatographic setups [IV], Figure 9, but the author

Industrial Emissions Directive, supplemented by horizontal legislation (e.g., Framework Directives on Waste and Water, Emissions Trading System, etc) and guidance on operating