A laser microdissection-based workflow for FFPE tissue microproteomics: Important considerations for small sample processing.

Proteomic methods are today widely applied to formalin-fixed paraffin-embedded (FFPE) tissue samples for several applications in research, especially in molecular pathology. To date, there is an unmet need for the analysis of small tissue samples, such as for early cancerous lesions. Indeed, no method has yet been proposed for the reproducible processing of small FFPE tissue samples to allow biomarker discovery. In this work, we tested several procedures to process laser microdissected tissue pieces bearing less than 3000 cells. Combined with appropriate settings for liquid chromatography mass spectrometry-mass spectrometry (LC-MS/MS) analysis, a citric acid antigen retrieval (CAAR)-based procedure was established, allowing to identify more than 1400 proteins from a single microdissected breast cancer tissue biopsy. This work demonstrates important considerations concerning the handling and processing of laser microdissected tissue samples of extremely limited size, in the process opening new perspectives in molecular pathology. A proof of the proposed method for biomarker discovery, with respect to these specific handling considerations, is illustrated using the differential proteomic analysis of invasive breast carcinoma of no special type and invasive lobular triple-negative breast cancer tissues. This work will be of utmost importance for early biomarker discovery or in support of matrix-assisted laser desorption/ionization (MALDI) imaging for microproteomics from small regions of interest.

Use of radiofrequency power to enable glow discharge optical emission spectroscopy ultrafast elemental mapping of combinatorial libraries with nonconductive components: nitrogen-based materials.

Combinatorial chemistry and high-throughput techniques are an efficient way of exploring optimal values of elemental composition. Optimal composition can result in high performance in a sequence of material synthesis and characterization. Materials combinatorial libraries are typically encountered in the form of a thin film composition gradient which is produced by simultaneous material deposition on a substrate from two or more sources that are spatially separated and chemically different. Fast spatially resolved techniques are needed to characterize structure, composition, and relevant properties of these combinatorial screening samples. In this work, the capability of a glow discharge optical emission spectroscopy (GD-OES) elemental mapping system is extended to nitrogen-based combinatorial libraries with nonconductive components through the use of pulsed radiofrequency power. The effects of operating parameters of the glow discharge and detection system on the achievable spatial resolution were investigated as it is the first time that an rf source is coupled to a setup featuring a push-broom hyperspectral imaging system and a restrictive anode tube GD source. Spatial-resolution optimized conditions were then used to characterize an aluminum nitride/chromium nitride thin-film composition spread. Qualitative elemental maps could be obtained within 16.8 s, orders of magnitude faster than typical techniques. The use of certified reference materials allowed quantitative elemental analysis maps to be extracted from the emission intensity images. Moreover, the quantitative procedure allowed correcting for the inherent emission intensity inhomogeneity in GD-OES. The results are compared to quantitative depth profiles obtained with a commercial GD-OES instrument.

Microproteomic Profiling of High-Grade Squamous Intraepithelial Lesion of the Cervix: Insight into Biological Mechanisms of Dysplasia and New Potential Diagnostic Markers.

PURPOSE: High-grade squamous intraepithelial lesion (HSIL) is a known precursor for squamous cell carcinoma of uterine cervix. Although it is known that SILs are associated to infection by human papillomavirus, downstream biological mechanisms are still poorly described. In this study, we compared the microproteomic profile of HSIL to normal tissues: ectocervix (ectoC) and endocervix (endoC). EXPERIMENTAL DESIGN: Tissue regions of endoC, ectoC, and HSlL were collected by laser microdissection (3500 cells each) from five patients. Samples were processed and analyzed using our recently developed laser microdissection-based microproteomic method. Tissues were compared in order to retrieve HSIL's proteomic profile. Potentially interesting proteins for pathology were stained by immunohistochemistry. RESULTS: We identified 3072 proteins among the fifteen samples and 2386 were quantified in at least four out of the five biological replicates of at least one tissue type. We found 236 proteins more abundant in HSIL. Gene ontology enrichments revealed mechanisms of DNA replication and RNA splicing. Despite the squamous nature of HSIL, a common signature between HSIL and endoC could be found. Finally, potential new markers could support diagnosis of dysplasia in SILs. CONCLUSION AND CLINICAL RELEVANCE: This microproteomic investigation of HSIL gives insights into the biology of cervical precancerous lesions.

MALDI Imaging Combined with Laser Microdissection-Based Microproteomics for Protein Identification: Application to Intratumor Heterogeneity Studies.

Matrix-assisted laser desorption ionization (MALDI) imaging is widely used for in situ proteomic mapping and finds multiple applications in pathology. However, low fragmentation yields in MALDI avoid an optimal identification of peptides from tissues. On the other hand, LMD-based microproteomic analyses allow for the identification of hundreds to thousands of proteins from small tissue regions. Herein, we present the combination of MALDI imaging and LMD-based microproteomic approaches for parallel identification. We illustrate the workflow with an application to intratumor heterogeneity studies.

MALDI Imaging-Guided Microproteomic Analyses of Heterogeneous Breast Tumors-A Pilot Study.

Matrix-assisted laser desorption/ionization (MALDI) imaging is an ideal tool to study intratumor heterogeneity (ITH) and its implication in prognostic stratification of patients. However, there are some drawbacks concerning protein identification. On the other hand, laser microdissection (LMD)-based microproteomics allows retrieving thousands of protein identifications from small tissue pieces. As a proof of concept, the authors combine these two complementary approaches to analyze heterogeneous regions in breast tumors. Invasive ductal breast cancer FFPE tissue sections from five patients are analyzed by MALDI imaging and the dataset is processed by segmentation. Heterogeneous regions within tumors are processed by LMD-based microproteomics, in duplicates. Liquid chromatography-tandem mass spectrometry data are classified by hierarchical clustering. Heterogeneous tissue regions are discriminated on the basis of their actual molecular heterogeneity. The dataset is correlated with MALDI imaging to identify m/z values discriminating heterogeneous regions. The molecular characterization of cell clones in tumors related to bad patient outcome could have great impact for pathology. A combined application of LMD-based microproteomics and MALDI imaging for ITH studies is presented.

An Improved Molecular Histology Method for Ion Suppression Monitoring and Quantification of Phosphatidyl Cholines During MALDI MSI Lipidomics Analyses.

Tissue lipidomics is one of the latest omics approaches for biomarker discovery in pharmacology, pathology, and the life sciences at large. In this context, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is the most versatile tool to map compounds within tissue sections. However, ion suppression events occurring during MALDI MSI analyses make it impossible to use this method for quantitative investigations without additional validation steps. This is especially true for lipidomics, since different lipid classes are responsible for important ion suppression events. We propose here an improved lipidomics method to assess local ion suppression of phospatidylcholines in tissues. Serial tissue sections were spiked with different amounts of PC(16:0 d31/18:1) using a nebulization device. Settings for standard nebulization were strictly controlled for a detection similar to when using spiked tissue homogenates. The sections were simultaneously analyzed by MALDI MSI using a Fourier transform ion cyclotron resonance analyzer. Such a spray-based approach allows taking into account the biochemical heterogeneity of the tissue for the detection of PC(16:0 d31/18:1). Thus, here we present the perspective to use this method for quantification purposes. The linear regression lines are considered as calibration curves and we calculate PC(16:0/18:1) quantification values for different ROIs. Although those values need to be validated by a using a different independent approach, the workflow offers an insight into new quantitative mass spectrometry imaging (q-MSI) methods. This approach of ion suppression monitoring of phosphocholines in tissues may be highly interesting for a large range of applications in MALDI MSI, particularly for pathology using translational science workflows.

Endogenous and exogenous hydrogen influence on amorphous silicon thin films analysis by pulsed radiofrequency glow discharge optical emission spectrometry.

During the last decade the photovoltaic industry has been growing rapidly. One major strategy to reduce the production costs is the use of thin film solar cells based on hydrogenated amorphous silicon (a-Si:H). The potential of pulsed radiofrequency glow discharge coupled to optical emission spectrometry (rf-PGD-OES) for the analysis of such type of materials has been investigated in this work. It is known that when hydrogen is present in the argon discharge, even in small quantities, significant changes can occur in the emission intensities and sputtering rates measured. Therefore, a critical comparison has been carried out by rf-PGD-OES, in terms of emission intensities, penetration rates and depth resolution for two modes of hydrogen introduction in the discharge, manually external hydrogen in gaseous form (0.2% H(2)-Ar) or internal hydrogen, sputtered as a sample constituent. First, a comparative optimisation study (at 600 Pa and 50 W) was performed on conducting materials and on a silicon wafer varying the pulse parameters: pulse frequency (500 Hz-20 kHz) and duty cycle (12.5-50%). Finally, 600 Pa, 50 W, 10 kHz and 25% duty cycle were selected as the optimum conditions to analyse three types of hydrogenated samples: an intrinsic, a B-doped and a P-doped layer based on a-Si:H. Enhanced emission intensities have been measured for most elements in the presence of hydrogen (especially for silicon) despite the observed reduced sputtering rate. The influence of externally added hydrogen and that of hydrogen sputtered as sample constituent from the analysed samples has been evaluated.

Depth profile characterization of Zn-TiO2 nanocomposite films by pulsed radiofrequency glow discharge-optical emission spectrometry.

In recent years particular effort is being devoted towards the development of radiofrequency (rf) pulsed glow discharges (GDs) coupled to optical emission spectrometry (OES) for depth profile analysis of materials with technological interest. In this work, pulsed rf-GD-OES is investigated for the fast and sensitive depth characterization of Zn-TiO(2) nanocomposite films deposited on conductive substrates (Ti and steel). The first part of this work focuses on assessing the advantages of pulsed GDs, in comparison with the continuous GD, in terms of analytical emission intensities and emission yields. Next, the capability of pulsed rf-GD-OES for determination of thickness and compositional depth profiles is demonstrated by resorting to a simple multi-matrix calibration procedure. A rf forward power of 75 W, a pressure of 600 Pa, 10 kHz pulse frequency and 50% duty cycle were selected as GD operation parameters.Quantitative depth profiles obtained with the GD proposed methodology for Zn-TiO(2) nanocomposite films, prepared by the occlusion electrodeposition method using pulsed reverse current electrolysis, have proved to be in good agreement with results achieved by complementary techniques, including scanning electron microscopy and inductively coupled plasma-mass spectrometry. The work carried out demonstrates that pulsed rf-GD-OES is a promising tool for the fast analytical characterization of nanocomposite films.