Coşkun Güzel

Sample preparation issues for tissue imaging by imaging MS.

Imaging MS is a powerful technique that combines the chemical and spatial analysis of surfaces. It allows spatial localization of multiple different compounds that are recorded in parallel without the need of a label. It is currently one of the rapidly developing techniques in the proteomics toolbox. Different complementary imaging MS methods, i.e. MALDI and secondary ion MS imaging for direct tissue analysis, can be applied on exactly the same tissue sample. This allows the identification of small molecules, peptides and proteins present on the same sample surface. Sample preparation is crucial to obtain high quality, reliable and reproducible complementary molecular images. It is essential to optimize the conditions for each step in the sample preparation protocol, ranging from sample collection and storage to surface modification. In this article, we review and discuss the importance of correct sample treatment in case of MALDI and secondary ion MS imaging experiments and describe the experimental requirements for optimal sample preparation.

Multiple reaction monitoring assay for pre-eclampsia related calcyclin peptides in formalin fixed paraffin embedded placenta

Although the cause of pre-eclampsia during pregnancy has not been elucidated yet, it is evident that placental and maternal endothelial dysfunction is involved. We previously demonstrated that in early onset pre-eclampsia placental calcyclin (S100A6) expression is significantly higher compared to controls ( De Groot , C. J. ; Clin. Proteomics 2007 , 1 , 325 ). In the current study, the results were confirmed and relatively quantified by using multiple reaction monitoring (MRM) on two peptide fragments of calcyclin. Cells were obtained from control (n = 5) and pre-eclamptic placental (n = 5) tissue collected by laser capture microdissection from formalin-fixed paraffin-embedded (FFPE) material treated with a solution to reverse formalin fixation. Two calcyclin peptides with an extra glycine inserted in the middle of the amino acid sequence were synthesized and used as an internal reference. Data presented show that MRM on laser microdissected material from FFPE tissue material is possible. The developed MRM assay to study quantitative levels of proteins in FFPE laser microdissected cells using nonisotopic-labeled chemical analogs of mass tagged internal references showed that in pre-eclamptic patients elevated levels of calcyclin is observed in placental trophoblast cells compared to normal trophoblast cells. By immunohistochemistry, we were able to confirm this observation in a qualitative manner.

Reproducibility of protein identification of selected cell types in Barrett's esophagus analyzed by combining laser-capture microdissection and mass spectrometry.

Barretts esophagus (BE) is associated with increased risk of esophageal adenocarcinoma (EAC) and characterized by replacement of normal esophageal squamous epithelium by columnar epithelium. These alterations are also reflected in changes in the protein-expression profiles of the cell types involved. To separately investigate the proteomes of selected cell-types we combined laser-capture microdissection (LCM) and liquid chromatography-mass spectrometry (LC-MS). Aims were to determine the sensitivity, specificity, and technical reproducibility of the sampling method, and the biological variability within and between biopsies and patients. Frozen biopsies were cryo-sectioned, samples of around 2000 epithelial or stroma cells microdissected, digested and measured by Orbitrap LC-MS. Proteins were then identified by MS/MS database search and quantified by label-free analysis. An average of 366 protein-groups were identified per sample, and more protein-groups were found in epithelial samples than in stromal samples (442 vs 301, p < 0.0001). Altogether, 1254 distinct protein-groups were found, 289 and 88 of them significantly more often in epithelial and stroma samples, respectively. We assessed five different types of reproducibilities (run-to-run, intrabiopsy, biopsy-to-biopsy, experiment-to-experiment, and patient-to-patient) for protein identification and protein quantification. Reproducibility of protein identification ranged from 78 to 57%, and standard deviation of protein quantification was on patient-to-patient level four times higher than for run-to-run. We conclude that sampling around 2000 cells requires groups of 32 samples to detect significant, over 10-fold differences in protein abundances and thus creates a successful compromise between throughput and quality of results. We therefore believe that this method is suitable for investigating protein-expression profiles during carcinogenesis.

Specific peptides identified by mass spectrometry in placental tissue from pregnancies complicated by early onset preeclampsia attained by laser capture dissection

Preeclampsia is a common pregnancy‐specific syndrome that is diagnosed by the appearance of both increased blood pressure and proteinuria. Preeclampsia is associated with significant fetal and maternal morbidity and mortality. Although the etiology of preeclampsia is unknown, it is evident that abnormal placentation and trophoblast metabolism plays an important role. We therefore analyzed, identified, and verified specific proteins of villous trophoblast and villous stroma in small numbers of microdissected cells (approximately 125 cells) from seven placentas of women with pregnancies complicated by preeclampsia (cases) and seven uncomplicated pregnancies (controls). Tryptic peptide profiling by MALDI‐TOF MS was used for comparison and identification of significantly expressed peptides. The data were analyzed by ClinProTools (Bruker Daltonics) and by principal component analysis. Subsequently, a subset of placental tissues were homogenized and separated on a NanoLC system to obtain sequencing information (MS/MS spectra).

Trophoblast calcyclin is elevated in placental tissue from patients with early pre-eclampsia

The aetiology of pre-eclampsia is thought to originate from aberrant spiral artery remodelling and invasion evoking cellular oxidative stress. Previously, we discovered differentially expressed proteins in trophoblast cells of pre-eclamptic pregnancies. One of these proteins is calcyclin (S100A6); a Ca(2+)-binding protein associated with cellular stress response. By immunohistochemistry on formalin-fixed paraffin-embedded placental tissue, calcyclin expression was compared between women with early pre-eclampsia (n=72) and non-hypertensive control patients (n=66) (χ(2), p=0.006) blindly by two observers. Significantly more intense staining was seen in trophoblast cells of pre-eclamptic pregnancies compared to control placentas suggesting that trophoblast calcyclin is elevated in early pregnancy.

Elevated levels of protein AMBP in cerebrospinal fluid of women with preeclampsia compared to normotensive pregnant women

To investigate the cerebrospinal fluid (CSF) proteome of patients with preeclampsia (PE) and normotensive pregnant women, in order to provide a better understanding of brain involvement in PE.

Peptide fingerprinting of folate-responsive proteins in human B lymphoblasts and orofacial clefting

Eur J Clin Invest 2012; 42 (7): 738–750

Comparison of Targeted Mass Spectrometry Techniques With an Immunoassay: A Case Study For HSP90α

The objective of this study is to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay.

Proteomic alterations in early stage cervical cancer

Laser capture microdissection (LCM) allows the capture of cell types or well-defined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by “all-or-nothing” analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive “all-or-nothing” way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction Monitoring (PRM). Ingenuity Pathway Analysis using correction for multiple testing by permutation resulted in two networks that were differentially regulated in early stage cervical cancer compared with healthy tissue. From these networks, we learned that specific tumor mechanisms become effective during the early stage of cervical cancer.