RESUMEN
Proteins are essential agents of biological processes. To date, large-scale profiling of cell line collections including the Cancer Cell Line Encyclopedia (CCLE) has focused primarily on genetic information whereas deep interrogation of the proteome has remained out of reach. Here, we expand the CCLE through quantitative profiling of thousands of proteins by mass spectrometry across 375 cell lines from diverse lineages to reveal information undiscovered by DNA and RNA methods. We observe unexpected correlations within and between pathways that are largely absent from RNA. An analysis of microsatellite instable (MSI) cell lines reveals the dysregulation of specific protein complexes associated with surveillance of mutation and translation. These and other protein complexes were associated with sensitivity to knockdown of several different genes. These data in conjunction with the wider CCLE are a broad resource to explore cellular behavior and facilitate cancer research.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/metabolismo , Proteoma/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica/métodos , Humanos , Espectrometría de Masas/métodos , Inestabilidad de Microsatélites , Mutación/genética , Proteómica/métodosRESUMEN
The 2nd CASMS conference was held virtually through Gather. Town platform from October 17 to 21, 2022, with a total of 363 registrants including an outstanding and diverse group of scientists at the forefront of their research fields from both academia and industry worldwide, especially in the United States and China. The conference offered a 5-day agenda with an exciting scientific program consisting of two plenary lectures, 14 parallel symposia, and 4 special sessions in which a total of 97 invited speakers presented technological innovations and their applications in proteomics & biological mass spectrometry and metabo-lipidomics & pharmaceutical mass spectrometry. In addition, 18 invited speakers/panelists presented at 3 research-focused and 2 career development workshops. Moreover, 144 posters, 54 lightning talks, 5 sponsored workshops, and 14 exhibitions were presented, from which 20 posters and 8 lightning talks received presentation awards. Furthermore, the conference featured 1 MCP lectureship and 5 young investigator awardees for the first time to highlight outstanding mid-career and early-career rising stars in mass spectrometry from our society. The conference provided a unique scientific platform for young scientists (i.e., graduate students, postdocs and junior faculty/investigators) to present their research, meet with prominent scientists, and learn about career development and job opportunities (http://casms.org).
Asunto(s)
Espectrometría de Masas , Sociedades Científicas , Humanos , China , Preparaciones Farmacéuticas , Proteómica , Estados UnidosRESUMEN
Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.
Asunto(s)
Autofagia , Glutamina/metabolismo , Proteínas Quinasas/metabolismo , Sistema de Transporte de Aminoácidos ASC/metabolismo , Animales , Línea Celular Tumoral , Drosophila melanogaster , Humanos , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Complejos Multiproteicos , Proteínas , Serina-Treonina Quinasas TOR , Factores de Transcripción/metabolismoRESUMEN
Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of WT MYOC has remained elusive, and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum, and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution that is the equivalent of the pathological human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaperone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells, and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/metabolismo , Glaucoma/metabolismo , Glicoproteínas/metabolismo , Mutación Missense , Malla Trabecular/metabolismo , Cadena B de alfa-Cristalina/metabolismo , Sustitución de Aminoácidos , Animales , Cristalinas/genética , Cristalinas/metabolismo , Proteínas del Citoesqueleto/genética , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Femenino , Glaucoma/genética , Glaucoma/patología , Glicoproteínas/genética , Humanos , Masculino , Ratones Mutantes , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Ratas Sprague-Dawley , Malla Trabecular/patología , Cadena B de alfa-Cristalina/genéticaRESUMEN
Myocilin (MYOC) is a secreted protein found in human aqueous humor (AH) and mutations in the MYOC gene are the most common mutation observed in glaucoma patients. Human AH analyzed under non-reducing conditions suggests that MYOC is not normally found in a monomeric form, but rather is predominantly dimeric. Although MYOC was first reported almost 20 years ago, a technical challenge still faced by researchers is an inability to isolate full-length MYOC protein for experimental purposes. Herein we describe two methods by which to isolate sufficient quantities of human full-length MYOC protein from mammalian cells. One method involved identification of a cell line (HeLa S3) that would secrete full-length protein (15â¯mg/L) while the second method involved a purification approach from 293â¯cells requiring identification and modification of an internal MYOC cleavage site (Glu214/Leu215). MYOC protein yield from 293â¯cells was improved by mutation of two MYOC N-terminal cysteines (C47 and C61) to serines. Analytical size exclusion chromatography of our full-length MYOC protein purified from 293â¯cells indicated that it is predominantly dimeric and we propose a structure for the MYOC dimer. We hope that by providing methods to obtain MYOC protein, researchers will be able to utilize the protein to obtain new insights into MYOC biology. The ultimate goal of MYOC research is to better understand this target so we can help the patient that carries a MYOC mutation retain vision and maintain quality of life.
Asunto(s)
Humor Acuoso/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Ojo/química , Glicoproteínas/química , Multimerización de Proteína , Animales , Sitios de Unión/genética , Western Blotting , Células COS , Línea Celular , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Ojo/genética , Proteínas del Ojo/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
Epigenetic modifications of the genome, such as DNA methylation and posttranslational modifications of histone proteins, contribute to gene regulation. Growing evidence suggests that histone methyltransferases are associated with the development of various human diseases, including cancer, and are promising drug targets. High-quality generic assays will facilitate drug discovery efforts in this area. In this article, we present a liquid chromatography/mass spectrometry (LC/MS)-based S-adenosyl homocysteine (SAH) detection assay for histone methyltransferases (HMTs) and its applications in HMT drug discovery, including analyzing the activity of newly produced enzymes, developing and optimizing assays, performing focused compound library screens and orthogonal assays for hit confirmations, selectivity profiling against a panel of HMTs, and studying mode of action of select hits. This LC/MS-based generic assay has become a critical platform for our methyltransferase drug discovery efforts.
Asunto(s)
Cromatografía Liquida/métodos , Descubrimiento de Drogas/métodos , N-Metiltransferasa de Histona-Lisina/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Pruebas de Enzimas/métodos , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , S-Adenosilhomocisteína/análisis , S-Adenosilhomocisteína/metabolismoRESUMEN
Malignant tumors can evade destruction by the immune system by attracting immune-suppressive regulatory T cells (Treg) cells. The IKZF2 (Helios) transcription factor plays a crucial role in maintaining function and stability of Treg cells, and IKZF2 deficiency reduces tumor growth in mice. Here we report the discovery of NVP-DKY709, a selective molecular glue degrader of IKZF2 that spares IKZF1/3. We describe the recruitment-guided medicinal chemistry campaign leading to NVP-DKY709 that redirected the degradation selectivity of cereblon (CRBN) binders from IKZF1 toward IKZF2. Selectivity of NVP-DKY709 for IKZF2 was rationalized by analyzing the DDB1:CRBN:NVP-DKY709:IKZF2(ZF2 or ZF2-3) ternary complex X-ray structures. Exposure to NVP-DKY709 reduced the suppressive activity of human Treg cells and rescued cytokine production in exhausted T-effector cells. In vivo, treatment with NVP-DKY709 delayed tumor growth in mice with a humanized immune system and enhanced immunization responses in cynomolgus monkeys. NVP-DKY709 is being investigated in the clinic as an immune-enhancing agent for cancer immunotherapy.
Asunto(s)
Neoplasias , Factores de Transcripción , Animales , Humanos , Ratones , Factor de Transcripción Ikaros , Inmunoterapia , Neoplasias/terapia , Neoplasias/metabolismo , Linfocitos T Reguladores/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Bmi-1 and Mel-18 are structural homologues that belong to the Polycomb group of transcriptional regulators and are believed to stably maintain repression of gene expression by altering the state of chromatin at specific promoters. While a number of clinical and experimental observations have implicated Bmi-1 in human tumorigenesis, the role of Mel-18 in cancer cell growth has not been investigated. We report here that short hairpin RNA-mediated knockdown of either Bmi-1 or Mel-18 in human medulloblastoma DAOY cells results in the inhibition of proliferation, loss of clonogenic survival, anchorage-independent growth, and suppression of tumor formation in nude mice. Furthermore, overexpression of both Bmi-1 and Mel-18 significantly increases the clonogenic survival of Rat1 fibroblasts. In contrast, stable downregulation of Bmi-1 or Mel-18 alone does not affect the growth of normal human WI38 fibroblasts. Proteomics-based characterization of Bmi-1 and Mel-18 protein complexes isolated from cancer cells revealed substantial similarities in their respective compositions. Finally, gene expression analysis identified a number of cancer-relevant pathways that may be controlled by Bmi-1 and Mel-18 and also showed that these Polycomb proteins regulate a set of common gene targets. Taken together, these results suggest that Bmi-1 and Mel-18 may have overlapping functions in cancer cell growth.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Meduloblastoma/patología , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Homología de Secuencia de Aminoácido , Animales , Muerte Celular , Proliferación Celular , Supervivencia Celular , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Fibroblastos/citología , Expresión Génica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Meduloblastoma/genética , Ratones , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Proto-Oncogénicas/genética , ARN Interferente Pequeño/metabolismo , Ratas , Proteínas Represoras/genética , Trasplante HeterólogoRESUMEN
The C-Raf kinase is regulated by numerous phosphorylation steps. To quantify the most prominent phosphorylation sites of C-Raf, we performed mass spectrometry analysis of wild-type C-Raf and the constitutively active C-Raf mutant C-Raf-Y340D/Y341D. We confirmed phosphorylation of most of the sites reported in the literature with the exception that we did not detect phosphorylation of threonine 268/269 (autophosphorylation sites) and threonine 491/serine 494 (kinase activation loop). Importantly, we detected novel phosphorylation sites at the positions of serine 296 and 301. The degree of phosphorylation in these positions depends on the level of activation of C-Raf. Furthermore, we show here, using point mutant forms of C-Raf kinases with serine to alanine and serine to aspartic acid substitution, that serines 296 and 301 contribute to negative regulation of C-Raf.
Asunto(s)
Proteínas Proto-Oncogénicas c-raf/química , Proteínas Proto-Oncogénicas c-raf/metabolismo , Serina/metabolismo , Secuencia de Aminoácidos , Animales , Células COS , Dominio Catalítico/genética , Chlorocebus aethiops , Humanos , Datos de Secuencia Molecular , Mutación/genética , Mapeo Peptídico , Fosforilación , Proteínas Proto-Oncogénicas c-raf/genética , Alineación de Secuencia , Serina/genéticaRESUMEN
Comparative proteomic studies can lead to the identification of protein markers for disease diagnostics and protein targets for potential disease interventions. An inverse labeling strategy based on the principle of protein stable isotope labeling and mass spectrometric detection has been successfully applied to three general protein labeling methods. In contrast to the conventional single experiment approach, two labeling experiments are performed in which the initial labeling is reversed in the second experiment. Signals from differentially expressed proteins will distinguish themselves by exhibiting a characteristic pattern of isotope intensity profile reversal that will lead to the rapid identification of these proteins. Application of the inverse labeling method is demonstrated using model systems for protein chemical labeling, protein proteolytic labeling, and protein metabolic labeling. The methodology has clear advantages which are illustrated in the various studies. The inverse labeling strategy permits quick focus on signals from differentially expressed proteins (markers/targets) and eliminates ambiguities caused by the dynamic range of detection. In addition, the inverse labeling approach enables the unambiguous detection of covalent changes of proteins responding to a perturbation.
Asunto(s)
Espectrometría de Masas/métodos , Proteínas/química , Cromatografía Liquida/métodos , Hidrólisis , Marcaje IsotópicoRESUMEN
ABL1 tyrosine-kinase inhibitors (TKI) are front-line therapy for chronic myelogenous leukaemia and are among the best-known examples of targeted cancer therapeutics. However, the dynamic uptake into cells of TKIs of low molecular weight and their intracellular behaviour is unknown because of the difficulty of observing non-fluorescent small molecules at subcellular resolution. Here we report the direct label-free visualization and quantification of two TKI drugs (imatinib and nilotinib) inside living cells using hyperspectral stimulated Raman scattering imaging. Concentrations of both drugs were enriched over 1,000-fold in lysosomes as a result of their lysosomotropic properties. In addition, low solubility appeared to contribute significantly to the surprisingly large accumulation of nilotinib. We further show that the lysosomal trapping of imatinib was reduced more than tenfold when chloroquine is used simultaneously, which suggests that chloroquine may increase the efficacy of TKIs through lysosome-mediated drug-drug interaction in addition to the commonly proposed autophagy-inhibition mechanism.
Asunto(s)
Rastreo Celular/métodos , Células Eucariotas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Espectrometría Raman/métodos , Células Eucariotas/citología , Humanos , Microscopía Confocal , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The applicability of liquid chromatography-mass spectrometry (LC/MS) is often limited by throughput. The sharing of a mass spectrometer with multiple LCs significantly improves throughput; however, the reported systems have not been designed to fully utilize the MS duty cycle, and as a result to achieve maximum throughput. To fully utilize the mass spectrometer, the number of LC units that a MS will need to recruit is application dependent and could be significantly larger than the current commercial or published implementations. For the example of a single analyte, the number may approach the peak capacity to a first degree approximation. Here, the construction of a MS system that flexibly recruits any number of LC units demanded by the application is discussed, followed by the method to port a previously developed LC/MS method to the system to fully utilize a mass spectrometer. To demonstrate the performance and operation, a prototypical MS system of eight LC units was constructed. When 1-min chromatographic separations were performed in parallel on the eight LCs of the system, the average LC/MS analysis time per sample was 10.5 s when applied to the analysis of samples in 384-well plate format. This system has been successfully used to conduct large-volume biochemical assays with the analysis of a variety of molecular entities in support of drug discovery efforts. Allowing the recruitment of the number of LC units appropriate for a given application, this system has the potential to be a plug-and-play system to fully utilize a mass spectrometer.
Asunto(s)
Cromatografía Liquida/instrumentación , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Animales , Bovinos , Diglicéridos/química , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Hidrocortisona/análogos & derivados , Hidrocortisona/química , Modelos Químicos , Albúmina Sérica Bovina/química , Agua/químicaRESUMEN
Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.
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Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacología , Espectrometría de Masas/métodos , Cromatografía Liquida , Diacilglicerol O-Acetiltransferasa/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Estándares de Referencia , Reproducibilidad de los Resultados , Solventes/química , Factores de Tiempo , VolumetríaRESUMEN
High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.
Asunto(s)
Técnicas Químicas Combinatorias , Preparaciones Farmacéuticas/análisis , Espectrometría de Masas en Tándem/métodos , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Extracción en Fase Sólida/instrumentación , Extracción en Fase Sólida/métodos , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Tyrosine phosphorylation is a type of post-translational modification that plays a crucial role in signal transduction. Thus, the study of this modification at the proteomic level has great biological significance. However, because of the low abundance of tyrosine-phosphorylated proteins in total cell lysate, it is difficult to evaluate the dynamics of tyrosine phosphorylation at a global level. In this work, proteins carrying phosphotyrosine (pTyr) were first purified from whole cell lysate by immunoprecipitation using anti-pTyr monoclonal antibodies. After tryptic digestion, phosphopeptides were further enriched by IMAC and analyzed by LC-MS. Quantitative changes of tyrosine phosphorylation at the global level were evaluated using isotopic labeling (introduced at the methyl esterification step prior to IMAC). Using this double enrichment approach, we characterized interferon alpha (IFNalpha)-induced pTyr proteomic changes in Jurkat cells. We observed induced phosphorylation on several well documented as well as novel tyrosine phosphorylation sites on proteins involved in IFNalpha signal transduction, such as Tyk2, JAK1, and IFNAR subunits. A specific site on alpha-tubulin (Tyr-271) was observed to be phosphorylated upon treatment as well. Furthermore, our results suggest that LOC257106, a CDC42 GAP-like protein, is potentially involved in this pathway.
Asunto(s)
Antineoplásicos/farmacología , Cromatografía de Afinidad , Interferón-alfa/farmacología , Fosfotirosina/metabolismo , Proteómica , Receptores de Interferón/metabolismo , Transducción de Señal , Cromatografía Liquida , Humanos , Inmunoprecipitación , Janus Quinasa 1 , Células Jurkat , Metales , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/metabolismo , Receptores de Interferón/clasificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , TYK2 Quinasa , Tirosina/metabolismoRESUMEN
The inverse labeling/mass spectrometry strategy has been applied to protein metabolic (15)N labeling for gel-free proteomics to achieve the rapid identification of protein markers/targets. Inverse labeling involves culturing both the perturbed (by disease or by a drug treatment) and control samples each in two separate pools of normal and (15)N-enriched culture media such that four pools are produced as opposed to two in a conventional labeling approach. The inverse labeling is then achieved by combining the normal (14)N-control with the (15)N-perturbed sample, and the (15)N-control with the (14)N-perturbed sample. Both mixtures are then proteolyzed and analyzed by mass spectrometry (coupled with on-line or off-line separation). Inverse labeling overcomes difficulties associated with protein metabolic labeling with regard to isotopic peak correlation and data interpretation in the single-experiment approach (due to the non-predictable/variable mass difference). When two data sets from inverse labeling are compared, proteins of differential expression are readily recognized by a characteristic inverse labeling pattern or apparent qualitative mass shifts between the two inverse labeling analyses. MS/MS fragmentation data provide further confirmation and are subsequently used to search protein databases for protein identification. The methodology has been applied successfully to two model systems in this study. Utilizing the inverse labeling strategy, one can use any mass spectrometer of standard unit resolution, and acquire only the minimum, essential data to achieve the rapid and unambiguous identification of differentially expressed protein markers/targets. The strategy permits quick focus on the signals of differentially expressed proteins. It eliminates the detection ambiguities caused by the dynamic range of detection. Finally, inverse labeling enables the detection of covalent changes of proteins responding to a perturbation that one might fail to distinguish with a conventional labeling experiment.