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PubMed2003

Mass spectrometry-based proteomics.

Recent successes illustrate the role of mass spectrometry-based proteomics as an indispensable tool for molecular and cellular biology and for the emerging field of systems biology. These include the study of protein-protein interactions via affinity-based isolations on a small and proteome-wide scale, the mapping of numerous organelles, the concurrent description of the malaria parasite genome and proteome, and the generation of quantitative protein profiles from diverse species. The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.

Animals↗

PubMed2026

Distinct Effects of Rap1 Subtype A GTPase Deficiency on the Male Mouse Heart.

This study utilized a genetically engineered mouse model deficient in the small GTPase Rap1A (knockout/Rap1A-null) to understand the biological role of Rap1A in the heart. We examined differential protein expression in the left ventricle of Rap1A-null versus wild-type control C57BL/6 male mice (~5 months) using proteomics (nanoLC-MS/MS quantitative analysis), and in the whole heart of aged male mice (~16 months) using MAL-DI-TOF/TOF mass spectrometry. Additionally, we used an experimental model of acute cardiovascular stress and assessed the impact on heart tissue histology, gene expression and mortality risk. Rap1A-deficient hearts showed reduced size and reduced heart and left ventricular weights. Significantly reduced gene expression of extracellular matrix collagen type I and collagen type III was present under baseline and cardiovascular stress conditions. Assessment of the proteomic profile identified a crucial role of Rap1A in promoting healthy ventricular myocardium, as its deficiency exhibited increased impact on cytoskeletal, mitochondrial, metabolic and contractile protein expression in young and aged mice. In young Rap1A-deficient mice, overrepresentation analysis revealed markers myosin heavy chain 7 (β-MHC) and alpha-actinin-2 (α-actinin-2) associated with cardiomyopathies, and upon cardiac stress, showed mortality risk compared to controls. Altogether, these findings provide important insights into the role of Rap1A in cardiac structure and remodeling under basal and stress conditions in male mice.

Animals↗

PubMed2026

Proteome-wide Ubiquitinome Profiling Reveals Substrate-specific Dynamics Within the USP7 Network.

USP7 is a pleiotropic deubiquitylating enzyme that is involved in tumor suppression, (neuro) development, chromatin regulation and the DNA damage response. How USP7 regulates these diverse pathways is still unclear. Here, we report data-independent acquisition and label free quantitation mass spectrometry to profile the proteome-wide impact of USP7 on substrate de-ubiquitylation and overall protein abundance. First, we identified proteins associated with endogenous USP7 by immunopurification followed by data-independent acquisition and label free quantitation mass spectrometry. Integration of our new results with earlier interactomes of epitope-tagged USP7 yielded a consensus set of high-confidence protein targets. Domain mapping analysis revealed that, in addition to the TRAF domain, the ubiquitin-like domains of USP7 play a key role in substrate selection. Using specific enrichment of tryptic K-ε-GG peptides, we mapped proteome-wide changes in ubiquitinome dynamics following inhibition of USP7. Combining unbiased proteome-wide and targeted quantitative mass spectrometry revealed that deubiquitylation by USP7 can have different effects on the stability of distinct substrates, and suggests that USP7's activity profile is substrate-dependent rather than an intrinsic enzymatic property. Thus, in addition to providing a proteome-wide map of USP7 target sites, our multi-angle proteomics approach reveals that the effects of USP7-mediated deubiquitylation on its targets are remarkably variable and substrate-specific. Finally, based on these detailed molecular insights we show how USP7 connects various neurodevelopmental syndromes and tumor suppression pathways.

Ubiquitin-Specific Peptidase 7↗

PubMed1999

Proteome analysis. II. Protein subcellular redistribution: linking physiology to genomics via the proteome and separation technologies involved.

While annotated two-dimensional (2D) gel electrophoresis databases contain thousands of proteins, they do not represent the entire genome. High-molecular-mass proteins in particular are conspicuously absent from such databases. Filamin is prototypical of this class of proteins since it is a dimer with relative molecular mass (Mr) of 520000 containing at least 240 potential phosphorylation sites. Filamin is not readily separated by current 2D procedures, and is difficult to study with respect to cycles of phosphorylation-dephosphorylation. Novel technologies are needed to identify biochemical pathways impinging upon such targets. The success of immunofluorescence microscopy as a research tool can be attributed in part to the fact that proteins redistribute in response to a variety of physiological stimuli. Comparable quantitative methods are required in proteome analysis. Three components are necessary for development of an approach that is capable of screening for protein redistribution events: (1) subcellular fractionation, (2) protein labeling and (3) data acquisition. An integrated approach is presented that utilizes differential detergent fractionation combined with reversible, luminescent protein stains and analytical imaging for high-throughput analysis of signal transduction events leading to protein subcellular redistribution. The procedure has been successfully implemented to rapidly define key second messenger pathways leading to endothelial cell junctional permeability and to guide in the design of a new family of peptide-based anti-inflammatory drugs.

Amino Acid Sequence↗

PubMed2000

Sensitive immunoassay of tissue cell proteins procured by laser capture microdissection.

Coupling laser capture microdissection (LCM) with sensitive quantitative chemiluminescent immunoassays has broad applicability in the field of proteomics applied to normal, diseased, or genetically modified tissue. Quantitation of the number of prostate-specific antigen (PSA) molecules/cell was conducted on human prostate tissue cells procured by LCM from fixed and stained frozen sections. Under direct microscopic visualization, laser shots 30 microm in diameter captured specific cells from the heterogeneous tissue section onto a polymer transfer surface. The cellular macromolecules from the captured cells were solubilized in a microvolume of extraction buffer and directly assayed using an automated (1.5 hour) sandwich chemiluminescent immunoassay. Calibration of the chemiluminescent assay was conducted by developing a standard curve using known concentrations of PSA. After the sensitivity, precision, and linearity of the chemiluminescent assay was verified for known numbers of solubilized microdissected tissue cells, it was then possible to calculate the number of PSA molecules per microdissected tissue cell for case samples. In a study set of 20 cases, using 10 replicate samples of 100 laser shots per sample, the within-run (intraassay) SD was approximately 10% of the mean or less for all cases. In this series the number of PSA molecules per microdissected tissue cell ranged from 2 x 10(4) to 6. 3 x 10(6) in normal epithelium, prostate intraepithelial neoplasia (PIN), and invasive carcinoma. Immunohistochemical staining of human prostate for PSA was compared with the results of the soluble immunoassay for the same prostate tissue section. Independent qualitative scoring of anti-PSA immunohistochemical staining intensity paralleled the LCM quantitative immunoassay for each tissue subpopulation and verified the heterogeneity of PSA content between tissue subpopulations in the same case. Extraction buffers were successfully adapted for both secreted and membrane-bound proteins. This technology has broad applicability for the quantitation of protein molecules in pure populations of tissue cells.

Calibration↗

PubMed2001

Protein functions and biological contexts.

The availability of a rough draft of the predicted human proteome allows an evaluation of the extent to which the predicted and biochemical functions of proteins are in alignment, and the roles of different technologies and approaches to understanding human diseases and instantiating therapeutics. Microarray technologies at the transcriptomic and proteomic levels can be high throughput and excellent for diagnostic purposes, but their informational outputs are inferior in quality to those emerging from the co- and post-translational levels and from antibody-based molecular anatomy. It is now abundantly clear that data transfer between the transcriptome and proteome is not straightforward, and that increasing emphasis needs to be placed on pure proteomic approaches at the structural, quantitative, cell biological and phenomic levels, with special focus on embryogenic and foetal processes. Finally, the precision genetic engineering that is required to evaluate the functional significance of context-dependent protein interactions underpinned by post-translational modifications and proteolytic cleavage events, is still too time consuming and rudimentary to be implemented on a large scale in the mouse, and basic principles and first order networks will need to be sorted out in even simpler model systems such as Drosophila.

Databases, Protein↗

PubMed2005

A sensitive method for the quantitative measurement of protein thiol modification in response to oxidative stress.

The combination of proteomics with highly specific and sensitive affinity techniques is important for the identification of posttranslational modifications by reactive oxygen and nitrogen species (ROS/RNS). One of the most pressing problems with this approach is to determine accurately the extent of modification of specific amino acids, such as cysteine residues, in a complex protein sample. A number of techniques relevant to free radical biology use biotin tagging as a method to follow protein modification with high sensitivity and specificity. To realize the potential of this approach to provide quantitative data, we have prepared a series of biotinylated proteins through the modification of lysine residues. These proteins were then used as quantitative standards in electrophoretic separation of protein samples labeled with biotin-conjugated iodoacetamide. The utility of the approach was assessed by measuring modification of thiols in response to exposure to thiol oxidants, as well as the amount of protein adduct formation with a biotin-tagged electrophilic lipid. Furthermore, using a combination of native and biotin-tagged cytochrome c, this method was used to quantitate the amount of thiol relative to the amount of protein in a given spot on a two-dimensional gel. Thus, we have developed a versatile, cost-effective standard that can be used in proteomic methods to quantitate biotin tags in response to oxidative stress.

Animals↗

PubMed2005

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry.

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.

Anemia, Sickle Cell↗

PubMed2006

Shotgun proteomics using the iTRAQ isobaric tags.

Shotgun proteomic methods involving isobaric tagging of peptides enable high-throughput proteomic analysis. iTRAQ reagents allow simultaneous identification and quantitation of proteins in four different samples using tandem mass spectrometry (MS). In this article, we provide a brief description of proteome analysis using iTRAQ reagents and review the current applications of these reagents in proteomic studies. We also compare different aspects of protein identification including protein sequence coverage and proteome coverage obtained using iTRAQ reagents with those using other shotgun proteomic techniques. We briefly discuss the issue of isotope purity correction in measured peak areas during protein quantitation using iTRAQ reagents. Finally, we conclude with some of the current challenges in MS-based proteomic analysis that are limiting protein identifications obtained by different shotgun proteomic methods.

Animals↗

PubMed2004

Proteomic analysis to identify breast cancer biomarkers in nipple aspirate fluid.

PURPOSE: Proteomic analysis of breast nipple aspirate fluid (NAF) holds promise as a noninvasive method to identify markers of breast cancer. The objectives of the study were to: (a) describe the NAF proteome, (b) identify candidate markers of breast cancer in NAF by using proteomic analysis, and (c) validate the markers identified by using a quantitative, high-throughput ELISA analysis. EXPERIMENTAL DESIGN: For proteome analysis, NAF proteins from a single subject without breast cancer were separated by two-dimensional PAGE and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectometry identification. A total of 41 different proteins were identified, 25 of which were known to be secreted. To identify breast cancer markers, we separated 20 NAF samples (10 normal, 10 cancer) by two-dimensional PAGE. Three protein spots were detected that were up-regulated in three or more cancer samples. These spots were identified to be gross cystic disease fluid protein (GCDFP)-15, apolipoprotein D (apoD), and alpha1-acid glycoprotein (AAG). To validate these three potential biomarkers, 105 samples (53 from benign breasts and 52 from breasts with cancer) were analyzed using ELISA. RESULTS: Among all of the subjects, GCDFP-15 levels were lower (P <0.001) and AAG levels were higher (P=0.001) in breasts with cancer. This was also true in premenopausal (GCDFP-15, P=0.011; AAG, P=0.002) but not in postmenopausal women. GCDFP-15 levels were lowest (P=0.003) and AAG levels highest (P <0.001) in women with ductal carcinoma in situ (DCIS). Menopausal status influenced GCDFP-15 and AAG more in women without breast cancer than in women with breast cancer. apoD levels did not correlate significantly with breast cancer. CONCLUSIONS: Our study revealed that the NAF proteome, as defined by two-dimensional PAGE, consists of a limited number of proteins, and that the expression of AAG and GCDFP-15 correlates with disease presence and stage.

Adolescent↗

PubMed2026

Estimation of chloroplast macromolecular complex copy numbers and subunit stoichiometries during the Chlamydomonas reinhardtii cell cycle.

An unbiased, quantitative view of biomolecules in a living cell is a prerequisite for accurate modeling approaches and informs our understanding of cellular metabolism at scale. In this work, we used the total protein approach (TPA), in which the total protein mass of a given proteomics sample is used as a calibrator for absolute protein quantification, to determine protein abundances during the Chlamydomonas reinhardtii diurnal cycle. We use external, independently measured quantitative markers (metals, pigments) to assess the absolute protein abundances in unlabeled whole cell extracts. We calculate protein abundances in fg cell-1 of 7322 Chlamydomonas proteins, 2266 of which were captured in every time point, including the major proteins involved in the light reactions, photoprotection, proteostasis, and fatty acid metabolism during a cell cycle. As expected, Rubisco large and small subunits are present in a 1:1 stoichiometry, with the large subunit being the most abundant protein in our data set, averaging 5.05 × 106 molecules per cell, reflecting 2.7% of the total protein mass. We noticed that PSII is the most abundant complex involved in the light reactions with 2.08 × 106 complexes per cell. PSI averages 1.75 × 106 complexes per cell and cytochrome b6f averages 0.77 × 106 complexes per cell. The TPA is a robust tool to study proteome dynamics quantitatively, while avoiding artifacts due to biochemical fractionation. Our proteome data set with an unprecedented temporal resolution is a valuable resource to assess protein abundances during the cell cycle in the reference alga Chlamydomonas.

Chlamydomonas reinhardtii↗

PubMed2004

Advances in recombinant antibody microarrays.

Antibody microarrays, one emerging class of proteomic technologies, have broad applications in proteome analysis, disease diagnostics and quantitative analysis. Compared to DNA microarrays, protein targets have significantly more complex interactions with their ligands such as antibodies. To introduce antibody microarrays for clinical diagnostics and thus to complement or replace conventional immunoassays, several new developments are addressed. We discuss different microarray surfaces, immobilization techniques, detection systems and advantages and disadvantages of antibody microarrays compared to standard clinical techniques. Currently, the probes with highest specificity, well-characterized binding properties, and the possibility of large-scale production using display libraries are recombinant antibodies.

Animals↗

PubMed2001

Emerging technologies for large-scale screening of human tissues and fluids in the study of severe psychiatric disease.

Neuropsychiatric diseases such as schizophrenia and bipolar disorder are major causes of morbidity throughout the world. Despite extensive searches, no single gene, RNA transcript, or protein has been found which can, on its own, account for these disorders. Recently, the availability of genomic tools such as cDNA microarrays, serial analysis of gene expression (SAGE) and large-scale sequencing of cDNA libraries has allowed researchers to assay biological samples for a large number of RNA transcripts. Similarly, proteomic tools allow for the quantitation of a large number of peptides and proteins. These methods include two-dimensional electrophoresis and surface-enhanced laser desorption/ionization (SELDI). We have initiated experiments which apply these techniques to the comparison of RNAs and proteins expressed in clinical samples obtained from individuals with psychiatric diseases and controls. These methods have the potential to identify pathways that are involved in the pathogenesis of complex psychiatric disorders. The characterization of these pathways may allow for the development of new methods for the diagnosis and treatment of schizophrenia, bipolar disorder, and other human psychiatric diseases.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2002

Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins.

The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of beta-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from beta-casein were identified along with the phosphorylated peptides from alphas1-casein and alphas2-casein, known low-level (<5%) contaminants of commercially available beta-casein. All of the casein-phosphorylated residues identified by the present PhIAT approach correspond to previously documented sites of phosphorylation. The results illustrate the efficacy of the PhIAT-labeling strategy to not only enrich mixtures for phosphopeptides but also, more importantly, permit the detection and identification of low-level phosphopeptides. In addition, the differences in the phosphorylation state could be determined between phosphopeptides in comparative samples by stoichiometric conversion using the light and heavy isotopic versions of the PhIAT reagents. Overall, our results exemplify the application of the PhIAT approach and demonstrate its utility for proteome-wide phosphoprotein identification and quantitation.

Affinity Labels↗

PubMed2025

Temporal multiomics gene expression data of human embryonic stem cell-derived cardiomyocyte differentiation.

Human embryonic stem cells (hESCs) serve as a valuable in vitro model for studying early human developmental processes due to their ability to differentiate into all three germ layers. Here, we present a comprehensive multi-omics dataset generated by differentiating hESCs into cardiomyocytes via the mesodermal lineage, collecting samples at 10 distinct time points. We measured mRNA levels by mRNA sequencing (mRNA-seq), translation levels by ribosome profiling (Ribo-seq), and protein levels by quantitative mass spectrometry-based proteomics. Technical validation confirmed high quality and reproducibility across all datasets, with strong correlations between replicates. This extensive dataset provides critical insights into the complex regulatory mechanisms of cardiomyocyte differentiation and serves as a valuable resource for the research community, aiding in the exploration of mammalian development and gene regulation.

Humans↗

PubMed2025

Mapping the FOXA1 Interactome in ER+ Breast Cancer Cells Using Proximity Labeling Reveals Novel Interactions with the Orphan Nuclear Receptor NR2C2.

UNLABELLED: FOXA1 is a pioneer transcription factor essential for chromatin accessibility and transcriptional regulation in hormone-driven cancers. In breast cancer, FOXA1 plays a central role in facilitating nuclear receptor binding, reprogramming enhancer landscapes, and promoting transcriptional changes associated with therapy resistance. Whereas FOXA1's function has been primarily studied in the context of estrogen receptor-α (ER), its broader protein interaction network remains incompletely defined. In this study, we systematically map FOXA1-interacting proteins in ER-positive breast cancer cells using proximity-dependent biotin labeling (miniTurbo) combined with quantitative LC-MS/MS proteomics. We engineered MCF-7 cell lines stably expressing miniTurbo-tagged FOXA1 at either the N-terminus or C-terminus to ensure comprehensive coverage of interaction interfaces. This approach recovered known FOXA1 partners, including AR, MLL3, YAP1, and GATA3, and identified 157 previously unreported FOXA1 interactors. Notably, 42 of these novel partners, including NR2C2, were significantly associated with poor relapse-free survival in patients with ER-positive breast cancer. To demonstrate the utility of this resource, we characterized the FOXA1-NR2C2 interaction in depth. Integrating chromatin immunoprecipitation sequencing and RNA sequencing, we show that FOXA1 and NR2C2 co-occupy a subset of genomic regions and drive co-regulated transcriptional programs involved in tumor progression. Our study reveals an expanded FOXA1 interactome and new insights into its functional network in breast cancer, providing candidate proteins for further exploration as biomarkers or therapeutic targets. IMPLICATIONS: These findings expand the FOXA1 interactome in breast cancer and uncover new candidate proteins with potential as biomarkers and therapeutic targets in hormone-driven tumors.

Humans↗

PubMed2004

In vitro selection of ribozymes dependent on peptides for activity.

A peptide-dependent ribozyme ligase (aptazyme ligase) has been selected from a random sequence population based on the small L1 ligase. The aptazyme ligase is activated > 18,000-fold by its cognate peptide effector, the HIV-1 Rev arginine-rich motif (ARM), and specifically recognizes the Rev ARM relative to other peptides containing arginine-rich motifs. Moreover, the aptazyme ligase can preferentially recognize the Rev ARM in the context of the full-length HIV-1 Rev protein. The only cross-reactivity exhibited by the aptazyme is toward the Tat ARM. Reselection of peptide- and protein-dependent aptazymes from a partially randomized population yielded aptazymes that could readily discriminate against the Tat ARM. These results have important implications for the development of aptazymes that can be used in arrays for the detection and quantitation of multiple cellular proteins (proteome arrays).

Amino Acid Motifs↗

PubMed2006

New algorithm for 15N/14N quantitation with LC-ESI-MS using an LTQ-FT mass spectrometer.

A new algorithm (QN) for the (15)N /(14)N quantitation of relative protein abundances in complex proteomic samples is described. QN takes advantage of the high resolution, mass accuracy and throughput of the hybrid mass spectrometer LTQ-FT MS. Peptide quantitation is based on MS peak intensity (measured in the FT MS), while peptide identification is performed in the MS/MS mode (measured in the LTQ linear ion trap). Accuracy of the protein abundance is enhanced by a novel scoring procedure, allowing filtering of less reliable measurements of peptide abundances. The performance of QN is illustrated in the relative quantitative analysis of M. acetivorans C2A cultures grown with carbon monoxide vs methanol as substrate. Roughly 1,000 proteins were quantitated with an average CV of 9% for the protein abundance ratios. QN performs quantitation without manual intervention, does not require high processing power, and generates files compatible with the Guidelines for Proteomic Data Publication.

Algorithms↗