Iulia M. Lazar

Recent advances in the MS analysis of glycoproteins: Theoretical considerations.

Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large‐scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large‐scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in MS instrumentation and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow‐up to this work, we review innovative capillary and microfluidic‐MS workflows for the identification, sequencing and characterization of glycoconjugates.

MRM screening/biomarker discovery with linear ion trap MS: a library of human cancer-specific peptides.

BackgroundThe discovery of novel protein biomarkers is essential in the clinical setting to enable early disease diagnosis and increase survivability rates. To facilitate differential expression analysis and biomarker discovery, a variety of tandem mass spectrometry (MS/MS)-based protein profiling techniques have been developed. For achieving sensitive detection and accurate quantitation, targeted MS screening approaches, such as multiple reaction monitoring (MRM), have been implemented.MethodsMCF-7 breast cancer protein cellular extracts were analyzed by 2D-strong cation exchange (SCX)/reversed phase liquid chromatography (RPLC) separations interfaced to linear ion trap MS detection. MS data were interpreted with the Sequest-based Bioworks software (Thermo Electron). In-house developed Perl-scripts were used to calculate the spectral counts and the representative fragment ions for each peptide.ResultsIn this work, we report on the generation of a library of 9,677 peptides (p < 0.001), representing ~1,572 proteins from human breast cancer cells, that can be used for MRM/MS-based biomarker screening studies. For each protein, the library provides the number and sequence of detectable peptides, the charge state, the spectral count, the molecular weight, the parameters that characterize the quality of the tandem mass spectrum (p-value, DeltaM, Xcorr, DeltaCn, Sp, no. of matching a, b, yions in the spectrum), the retention time, and the top 10 most intense product ions that correspond to a given peptide. Only proteins identified by at least two spectral counts are listed. The experimental distribution of protein frequencies, as a function of molecular weight, closely matched the theoretical distribution of proteins in the human proteome, as provided in the SwissProt database. The amino acid sequence coverage of the identified proteins ranged from 0.04% to 98.3%. The highest-abundance proteins in the cellular extract had a molecular weight (MW)<50,000.ConclusionPreliminary experiments have demonstrated that putative biomarkers, that are not detectable by conventional data dependent MS acquisition methods in complex un-fractionated samples, can be reliable identified with the information provided in this library. Based on the spectral count, the quality of a tandem mass spectrum and the m/z values for a parent peptide and its most abundant daughter ions, MRM conditions can be selected to enable the detection of target peptides and proteins.

Recurrent chimeric fusion RNAs in non-cancer tissues and cells

Gene fusions and their products (RNA and protein) were once thought to be unique features to cancer. However, chimeric RNAs can also be found in normal cells. Here, we performed, curated and analyzed nearly 300 RNA-Seq libraries covering 30 different non-neoplastic human tissues and cells as well as 15 mouse tissues. A large number of fusion transcripts were found. Most fusions were detected only once, while 291 were seen in more than one sample. We focused on the recurrent fusions and performed RNA and protein level validations on a subset. We characterized these fusions based on various features of the fusions, and their parental genes. They tend to be expressed at higher levels relative to their parental genes than the non-recurrent ones. Over half of the recurrent fusions involve neighboring genes transcribing in the same direction. A few sequence motifs were found enriched close to the fusion junction sites. We performed functional analyses on a few widely expressed fusions, and found that silencing them resulted in dramatic reduction in normal cell growth and/or motility. Most chimeras use canonical splicing sites, thus are likely products of ‘intergenic splicing’. We also explored the implications of these non-pathological fusions in cancer and in evolution.

Recent advances in the MS analysis of glycoproteins: Capillary and microfluidic workflows

Recent developments in bioanalytical instrumentation, MS detection, and computational data analysis approaches have provided researchers with capabilities for interrogating the complex cellular glycoproteome, to help gain a better insight into the cellular and physiological processes that are associated with a disease and to facilitate the efforts centered on identifying disease‐specific biomarkers. This review describes the progress achieved in the characterization of protein glycosylation by using advanced capillary and microfluidic MS technologies. The major steps involved in large‐scale glycoproteomic analysis approaches are discussed, with special emphasis given to workflows that have evolved around complex MS detection functions. In addition, quantitative analysis strategies are assessed, and the bioinformatics aspects of glycoproteomic data processing are summarized. The developments in commercial and custom fabricated microfluidic front‐end platforms to ESI‐ and MALDI‐MS instrumentation, for addressing major challenges in carbohydrate analysis such as sensitivity, throughput, and ability to perform structural characterization, are further evaluated and illustrated with relevant examples.

General considerations for optimizing a capillary electrophoresis-electrospray ionization time-of-flight mass spectrometry system

Abstract Modern analytical instrumentation must be able to perform rapid, reliable, and sensitive analysis. The on-line combination of analytical techniques such as capillary electrophoresis (CE), electrospray ionization (ESI) and mass spectrometry (MS) can provide solutions to numerous problems related to complex mixtures of organic/inorganic, or biological materials. Optimum performance of complex instrumentation such as this can be achieved only if each individual component operates with maximum proficiency, and is in full harmony/compatibility with the other components. The present paper reports on the evaluation and optimization of a CE-ESI time-of-flight mass spectrometry (TOF-MS) system. The main features of the instrument are speed and sensitivity. Low amol (3–11) detection limits have been achieved with continuous infusion experiments, and the acquisition rate can be as high as 10 000 spectra s −1 . For CE–TOF-MS, minimum detection was in the very-low fmol (1–10) range. The major contributing factors to high quality analysis characteristic to each separate technique are considered, relevant examples are discussed, and fast, and sensitive analysis is demonstrated.

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17β-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was ∼60%–67% between duplicate, and ∼50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate ∼2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, ∼16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated.

Glycoproteomics on the rise: Established methods, advanced techniques, sophisticated biological applications

Glycosylation is the most complex form of protein PTMs. Affected proteins may carry dozens of glycosylation sites with tens to hundreds of glycan residues attached to every site. Glycosylated proteins have many important functions in biology, from cellular to organismal levels, being involved in cell–cell signaling, cell adhesion, immune response, host–pathogen interactions, and development and growth. Glycosylation, however, expands the biological functional diversity of proteins at the expense of a tremendous increase in structural heterogeneity. Aberrant glycosylation of cell surface proteins, as well as their detectable fingerprint in plasma samples, has been associated with cancer, inflammatory and degenerative diseases, and congenital disorders of glycosylation. Therefore, there are on‐going efforts directed toward developing new technologies and approaches for glycan sequencing and high‐throughput analysis of glycosylated proteins in complex samples with simultaneous characterization of both the protein and glycan moieties. This work is aimed primarily at pinpointing the challenges associated with the large‐scale analysis of glycoproteins and the latest developments in glycoproteomic research, with focus on recent advancements (2011–2012) in microcolumn separations and MS detection.

Evaluation of an electrospray interface for capillary electrophoresis-time-of-flight mass spectrometry

The electrospray technique has developed into a widely used ionization source for interfacing capillary electrophoresis (CE) to mass spectrometry (MS). However, its implementation is not always straightforward. A large number of factors have been found to be important contributors to the production of a high quality spray, and the efficient transfer of analytes from the CE column into the mass spectrometer can become, in certain cases, troublesome. An electrospray device which can accommodate operation with a liquid sheath, nebulizing gas and make-up gas, as well as operation without a liquid sheath in the microelectrospray mode, was constructed. The electrospray source was evaluated for efficient CE separations, which require the best performance.

Microfluidic chips for protein differential expression profiling

Biomarker discovery and screening using novel proteomic technologies is an area that is attracting increased attention in the biomedical community. Early detection of abnormal physiological conditions will be highly beneficial for diagnosing various diseases and increasing survivability rates. Clearly, progress in this area will depend on the development of fast, reliable, and highly sensitive and specific sample bioanalysis methods. Microfluidics has emerged as a technology that could become essential in proteomics research as it enables the integration of all sample preparation, separation, and detection steps, with the added benefit of enhanced sample throughput. The combination of these advantages with the sensitivity and capability of MS detection to deliver precise structural information makes microfluidics‐MS a very competitive technology for biomarker discovery. The integration of LC microchip devices with MS detection, and specifically their applicability to biomarker screening applications in MCF‐7 breast cancer cellular extracts is reported in this manuscript. Loading ∼0.1–1 μg of crude protein extract tryptic digest on the chip has typically resulted in the reliable identification of ∼40–100 proteins. The potential of an LC‐ESI‐MS chip for comparative proteomic analysis of isotopically labeled MCF‐7 breast cancer cell extracts is explored for the first time.

Capillary electrophoresis–time-of-flight mass spectrometry of drugs of abuse

The use of capillary electrophoresis (CE) for the determination of drugs of abuse was explored. A commercial CE system was interfaced with a laboratory-built time-of-flight mass spectrometer (TOFMS) which was equipped with a high-speed data acquisition system to provide accurate monitoring of efficient separations. Ionization of the CE eluent was achieved with an electrospray ionization source. Standard mixtures and seized samples were analyzed either by direct infusion of the analyte solutions or after separation by CE. Detection at the low femtomole level was obtained using CE-TOFMS.