Comparative Study of [Three] LC−MALDI Workflows for the Analysis of Complex Proteomic Samples

Hattan, Stephen J.; Marchese, Jason; Khainovski, Nikita; Martin, Steve; Juhász, Péter

doi:10.1021/pr050099e

Cited by 44 publications

(48 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous studies comparing MALDI and ESI instruments with respect to their sensitivity in proteomic analysis have come up with different results, but the detection levels of the two compared instruments mostly did not vary as strongly as in the present study (3,9,11,24,25). Here we have to point out that the speed of technological development in the two ionization branches has varied in recent years.…”

Section: Resultscontrasting

confidence: 62%

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Heßling

Büttner

Hecker

et al. 2013

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LCelectrospray ionization workflows.Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis.Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF-and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments. One-dimensional gel-based liquid chromatography mass spectrometry (GeLC-MS) 1 is a well-established technique in life science. In combination with in vivo labeling approaches such as stable isotope labeling by amino acids in cell culture (SILAC) (1) or 15 N labeling (2), it allows the relative quantification of large numbers of proteins in a complex sample. Mass spectrometry (MS) measurements in such workflows are predominantly performed with electrospray ionization (ESI)-based mass spectrometers. Online coupling of a liquid chromatography (LC) system with fast MS spectra acquisition and high-mass-accuracy ESI instruments in conjunction with fractionation on both protein and peptide levels allows the analysis of very complex samples in a relatively short period of time (3). The identification and quantification of data can be done in an automatic or semi-automatic manner with a variety of well-established software packages (4).In proteomic research, matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF) is mainly used for the analysis of non-complex protein samples that do not require fractionation on the peptide level by means of liquid chromatography. In particular, the analysis of single protein spots resulting from the twodimensional PAGE separation of complex samples is primarily carried out with this MS tec...

show abstract

Section: Resultscontrasting

confidence: 62%

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Heßling

Büttner

Hecker

et al. 2013

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

show abstract

“…Altogether, 1064 different proteins were identified in prediabetic replicates of which 854 (80.26%) were common to all three replicates. Controlling for the redundancy of identifications with the approach used by Hattan et al (19) and Zegels et al (20), the three technical replicates therefore yielded 2906 (964 ϩ 952 ϩ 990) identifications of which 2562 (854 ϫ 3) were found in all three replicates. The percentage of shared versus the total number of identifications was 88.16% (2562 of 2906); the percentage of identifications by only one replicate versus the total number of identifications was 2.62% ((23 ϩ 22 ϩ 31)/2906).…”

Section: Survey Of Gk Rat Liver Mitochondrial Proteome and Posttranslmentioning

confidence: 96%

Proteome, Phosphoproteome, and Hydroxyproteome of Liver Mitochondria in Diabetic Rats at Early Pathogenic Stages

Deng

Nie

Dai

et al. 2010

Molecular & Cellular Proteomics

100

View full text Add to dashboard Cite

It has been proposed that mitochondrial dysfunction is involved in the pathogenesis of type 2 diabetes (T2D). To dissect the underlying mechanisms, we performed a multiplexed proteomics study on liver mitochondria isolated from a spontaneous diabetic rat model before/after they were rendered diabetic. Altogether, we identified 1091 mitochondrial proteins, 228 phosphoproteins, and 355 hydroxyproteins. Mitochondrial proteins were found to undergo expression changes in a highly correlated fashion during T2D development. For example, proteins involved in ␤-oxidation, the tricarboxylic acid cycle, oxidative phosphorylation, and other bioenergetic processes were coordinately up-regulated, indicating that liver cells confronted T2D by increasing energy expenditure and activating pathways that rid themselves of the constitutively increased flux of glucose and lipid. Notably, activation of oxidative phosphorylation was immediately related to the overproduction of reactive oxygen species, which caused oxidative stress within the cells. Increased oxidative stress was also evidenced by our post-translational modification profiles such that mitochondrial proteins were more heavily hydroxylated during T2D development. Moreover, we observed a distinct depression of antiapoptosis and antioxidative stress proteins that might reflect a higher apoptotic index under the diabetic stage. We suggest that such changes in systematic metabolism were causally linked to the development of T2D. Comparing proteomics data against microarray data, we demonstrated that many T2D-related alterations were unidentifiable by either proteomics or genomics approaches alone, underscoring the importance of integrating different approaches. Our compendium could help to unveil pathogenic events in mitochondria leading to T2D and be useful for the discovery of diagnosis biomarker and therapeutic targets of T2D. Molecular & Cellular Proteomics 9:100 -116, 2010. Type 2 diabetes (T2D)1 has become a global health epidemic. It is recognized as a strong risk factor for cardiovascular diseases and for associated complications that result in substantial morbidity and mortality (1). Now it is recognized that T2D is a heterogeneous disease in which almost every aspect of the body's metabolism goes awry. The onset of T2D results from complex interactions between genetic and environmental factors. Several cellular dysfunctions and molecular defects are proposed to be associated with T2D, such as ␤-cell malfunction, impaired insulin secretion and function, chronic hyperglycemia, and other disturbances in systematic metabolism (1).Besides these findings, recently the topic of mitochondrial dysfunction has also gained a lot of attention in the T2D field (2-4). Numerous studies have revealed that various mitochondrial factors are of paramount importance in the pathogenesis of diabetes. For example, high fat diet-induced alterations in the mitochondrial compartment are associated with the development of insulin resistance and ectopic fat storage in liver (5). The dysfuncti...

show abstract

“…Ion-exchange chromatography is widely used in proteomics [170,171] and in metabolomics studies [172], for separations of oligosaccharides [173,174], and for quantification of proteins [175].…”

Section: Ion-exchange and Ion Chromatographymentioning

confidence: 99%

Polymethacrylate monolithic columns for capillary liquid chromatography

Urban

Jandera²

2008

J of Separation Science

121

View full text Add to dashboard Cite

In recent years, continuous separation media have attracted considerable attention because of the advantages they offer over packed columns. This research resulted in two useful monolithic material types, the first based on modified silica gel and the second on organic polymers. This work attempts to review advances in the development, characterization, and applications of monolithic columns based on synthetic polymers in capillary chromatography, with the main focus on monolithic beds prepared from methacrylate-ester based monomers. The polymerization conditions used in the production of polymethacrylate monolithic capillary columns are surveyed, with attention being paid to the concentrations of monomers, porogen solvents, and polymerization initiators as the system variables used to control the porous and hydrodynamic properties of the monolithic media. The simplicity of their preparation as well as the possibilities of controlling of their porous properties and surface chemistries are the main benefits of the polymer monolithic capillary columns in comparison to capillary columns packed with particulate materials. The application areas considered in this review concern mainly separations in reversed-phase chromatography, ion-exchange chromatography, hydrophobic and hydrophilic interaction modes; enzyme immobilization and sample preparation in the capillary chromatography format are also addressed.

show abstract

Comparative Study of [Three] LC−MALDI Workflows for the Analysis of Complex Proteomic Samples

Cited by 44 publications

References 49 publications

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Proteome, Phosphoproteome, and Hydroxyproteome of Liver Mitochondria in Diabetic Rats at Early Pathogenic Stages

Polymethacrylate monolithic columns for capillary liquid chromatography

Contact Info

Product

Resources

About