Stipendium China Scholarship Council

Title: Glycoproteomic Analysis of Cancer Tissues applying Picosecond Infrared Laser Extraction
Keywords: Analytical Biochemistry, Glycoproteome, Cancer, Picosecond Infrared Laser, Mass
Spectrometry
Background:
Professor Hartmut Schlüter’s research group, in the University Medical Center Hamburg-Eppendorf,
Hamburg, Germany, is a world-renowned team for its excellent work in the field of proteomics. His
group is focusing on clinical proteomics with state-of-the-art instruments, such as a picosecond
infrared laser (PIRL) system and hybrid orbitrap mass spectrometers (MS).
Prof. Schlüter’s group in collaboration with the group of Prof. Dwayne Miller (director of the Max
Planck Institute for the Structure and Dynamics of Matter (MPSD), Hamburg) has developed an
efficient and soft method for cold vaporization of tissues with PIRL, which has been published in the
Angew Chem Int Ed Engl. recently. In this publication it was demonstrated, that by the ablation of
tissue by irradiation with PIRL intact proteins are rapidly released without changing their structural
formula. Desorption of proteins from aqueous liquids by PIRL even did not abolish their enzymatic
activities. In addition, the protein yield extracted with PIRL from tissues, was about six times higher
than that from mechanical extraction methods. For proteomics this extraction method shows
significant advantages and promises to investigate proteomes in more detail.
I am a master student in mass spectrometry group at National Glycoengineering Research Center,
Shandong University, China. My research field focuses on the glycoproteomic characterization using
several analytical instruments including MS and HPLC. My knowledge and research experience
during master degree will make contributions to the future PhD study in University Medical Center
Hamburg-Eppendorf.
Scope of the research:
This PIRL extraction method has been confirmed to release intact proteins through desorption by
impulsive excitation (DIVE) from tissues. It is the aim of the PhD thesis to develop a novel
extraction method based on the PIRL-DIVE process for getting access to the intact glycoproteome
and then to apply the PIRL-DIVE glycoproteomics approach for the investigation of cancer tissues.
Therefore it is a further aim to identify cancer glycoprotein markers, which will promote early
diagnosis and therapy of cancer. The cancer tissue glycoproteome research covers a wide range of
disciplines including analytical biochemistry, clinical medicine and physical chemistry. The whole
study is designed into two steps.
In the first step, glycoproteins extracted by PIRL-DIVE are characterized by comprehensive
qualitative and quantitative analysis. Mouse liver tissues are chosen as model tissue samples and
liver CEACAM1 protein as the model glycoprotein, beside analysis of glycoproteomes. This part of
the study is performed as follows:
(1) PIRL-DIVE extraction of proteomes
Mouse liver tissues are irradiated by PIRL and the ablation plume is captured with a cryo-trap.
(2) Isolation and qualitative analysis of intact CEACAM1 proteins
CEACAM1, as one kind of highly glycosylated protein, had been extracted with PIRL by Prof.
Schlüter’s group, who observed that this glycoprotein survived the PIRL-DIVE process. Compared
with peptide bonds, glycan–peptide bonds are more fragile. In the future study it will be investigated,
whether there is loss of some parts of the glycan chains of CEACAM1 during the PIRL-DIVE
process. The extracted proteome and control CEACAM1 protein standard will be separated by
SDS-PAGE experiments. CEACAM1 will be transferred to blot membranes and the blot analyzed
with an anti-CEACAM1-antiserum P1 antibody. By diverse glycoproteomics methods, including
MALDI-MS as well as ESI-MS, the PIRL-ablated and isolated CEACAM1 will be comprehensively
analyzed and the results compared with results of the analysis of CEACAM1 yielded by classical
extraction methods.
(3) Qualitative and quantitative analysis of the glycoproteomes yielded by PIRL versus classical
extraction methods.
Mechanical extraction method and the PIRL-DIVE method will be applied to release glycoproteomes
from mouse tissues. Glycoproteins will be enriched and separated by SDS-PAGE and
isoelectric-focusing gels. Glycoproteins in all bands will be digested with trypsin without and with
prior removal of N-glycans enzymatically. The resulting migration profiles representing the relative
abundance of the gycoproteins will be compared. The comparison of the migration profiles will give
information about differences in the yield of intact glycoproteins with respect to the extraction
method as was shown in Kwiatkowski et al. 2016 (1).
It is assumed that by the first part of the study it can be demonstrated, that the glycoproteins obtained
by PIRL-DIVE process will remain intact, which proves the feasibility of this the PIRL extraction
method. In the second part of the study healthy and cancer tissues from the same organ (tonsils) of
individual patients will be analyzed. At least five patients will be investigated. This research is
performed as follows:
(1) Extraction of the glycoproteome
The PIRL-DIVE extraction of proteins will be applied to all the tissues. Glycoproteomes are
enriched with lectin-affinity chromatography.
(2) Characterization of glycoproteome
It has been confirmed that the glycoproteins from cancer tissues show different compositions with
respect to glycans, glycosylation sites and their abundances compared with healthy tissues. Therefore
the analysis of cancer glycoproteomes will mainly concentrate on three approaches. At the beginning
of each experiment, the enriched glycoproteins are digested by a protease. For analysis of glycans
enzymatic and chemical procedures will be used to release intact glycans and for permethylation of
the released glycans. Finally, MALDI-MS/MS will be used to analyze the structures of glycans. For
glycosylation site analysis, enzymatic processing is performed to release the glycans entirely (for
N-glycosylation sites) or partially (for O-glycosylation sites), leaving a site tag on the peptide. Then
LC-MS/MS is used to analyze the peptide mixture samples. Finally, the glycosylation sites are
1 Homogenization of human tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo
composition of protein species as compared to mechanical homogenization.
identified based on the mass shift of peptides using the algorithms like Mascot Distiller. The
iTRAQ-based technique will be applied for quantitative shotgun glycoproteomics.

Kwiatkowski M, Wurlitzer M, Krutilin A, Kiani P, Nimer R, Omidi M, Mannaa A, Bußmann T, Bartkowiak K, Kruber S, Uschold S, Steffen P, Lübberstedt J,
Küpker N, Petersen H, Knecht R, Hansen NO, Zarrine-Afsar A, Robertson WD, Miller RJ, Schlüter H. J Proteomics.
2016 Jan 8. pii: S1874-3919(16)30004-5.