Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

Quantitative Statistical Analysis of Standard and Human Blood Proteins from Liquid Chromatography, Electrospray Ionization, and Tandem Mass Spectrometry

It will be important to determine if the parent and fragment ion intensity results of liquid chromatography, electrospray ionization and tandem mass spectrometry (LC–ESI–MS/MS) experiments have been randomly and independently sampled from a normal population for the purpose of statistical analysis by general linear models and ANOVA. The tryptic parent peptide and fragment ion <i>m</i>/<i>z</i> and intensity data in the mascot generic files from LC–ESI–MS/MS of purified standard proteins, and human blood protein fractionated by partition chromatography, were parsed into a Structured Query Language (SQL) database and were matched with protein and peptide sequences provided by the X!TANDEM algorithm. The many parent and/or fragment ion intensity values were log transformed, tested for normality, and analyzed using the generic Statistical Analysis System (SAS). Transformation of both parent and fragment intensity values by logarithmic functions yielded intensity distributions that closely approximate the log-normal distribution. ANOVA models of the transformed parent and fragment intensity values showed significant effects of treatments, proteins, and peptides, as well as parent versus fragment ion types, with a low probability of false positive results. Transformed parent and fragment intensity values were compared over all sample treatments, proteins or peptides by the Tukey-Kramer Honestly Significant Difference (HSD) test. The approach provided a complete and quantitative statistical analysis of LC–ESI–MS/MS data from human blood

New Analysis Workflow for MALDI Imaging Mass Spectrometry: Application to the Discovery and Identification of Potential Markers of Childhood Absence Epilepsy

Childhood absence epilepsy is a prototypic form of generalized nonconvulsive epilepsy characterized by short impairments of consciousness concomitant with synchronous and bilateral spike-and-wave discharges in the electroencephalogram. For scientists in this field, the BS/Orl and BR/Orl mouse lines, derived from a genetic selection, constitute an original mouse model “in mirror” of absence epilepsy. The potential of MALDI imaging mass spectrometry (IMS) for the discovery of potential biomarkers is increasingly recognized. Interestingly, statistical analysis tools specifically adapted to IMS data sets and methods for the identification of detected proteins play an essential role. In this study, a new cross-classification comparative design using a combined discrete wavelet transformation-support vector machine classification was developed to discriminate spectra of brain sections of BS/Orl and BR/Orl mice. Nineteen <i>m</i>/<i>z</i> ratios were thus highlighted as potential markers with very high recognition rates (87–99%). Seven of these potential markers were identified using a top-down approach, in particular a fragment of Synapsin-I. This protein is yet suspected to be involved in epilepsy. Immunohistochemistry and Western Blot experiments confirmed the differential expression of Synapsin-I observed by IMS, thus tending to validate our approach. Functional assays are being performed to confirm the involvement of Synapsin-I in the mechanisms underlying childhood absence epilepsy

Exploring Three-Dimensional Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry Data: Three-Dimensional Spatial Segmentation of Mouse Kidney

Three-dimensional (3D) imaging has a significant impact on many challenges of life sciences. Three-dimensional matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an emerging label-free bioanalytical technique capturing the spatial distribution of hundreds of molecular compounds in 3D by providing a MALDI mass spectrum for each spatial point of a 3D sample. Currently, 3D MALDI-IMS cannot tap its full potential due to the lack efficient computational methods for constructing, processing, and visualizing large and complex 3D MALDI-IMS data. We present a new pipeline of efficient computational methods, which enables analysis and interpretation of a 3D MALDI-IMS data set. Construction of a MALDI-IMS data set was done according to the state-of-the-art protocols and involved sample preparation, spectra acquisition, spectra preprocessing, and registration of serial sections. For analysis and interpretation of 3D MALDI-IMS data, we applied the spatial segmentation approach which is well-accepted in analysis of two-dimensional (2D) MALDI-IMS data. In line with 2D data analysis, we used edge-preserving 3D image denoising prior to segmentation to reduce strong and chaotic spectrum-to-spectrum variation. For segmentation, we used an efficient clustering method, called bisecting <i>k</i>-means, which is optimized for hierarchical clustering of a large 3D MALDI-IMS data set. Using the proposed pipeline, we analyzed a central part of a mouse kidney using 33 serial sections of 3.5 μm thickness after the PAXgene tissue fixation and paraffin embedding. For each serial section, a 2D MALDI-IMS data set was acquired following the standard protocols with the high spatial resolution of 50 μm. Altogether, 512 495 mass spectra were acquired that corresponds to approximately 50 gigabytes of data. After registration of serial sections into a 3D data set, our computational pipeline allowed us to reveal the 3D kidney anatomical structure based on mass spectrometry data only. Finally, automated analysis discovered molecular masses colocalized with major anatomical regions. In the same way, the proposed pipeline can be used for analysis and interpretation of any 3D MALDI-IMS data set in particular of pathological cases