Absolute Quantification Labeling Research Articles

Mass Spectrometry-Based Proteomics for Relative Protein Quantification and Biomarker Identification in Primary Human Hepatocytes.

Liquid chromatography-tandem mass spectrometry-based proteomics is a highly sensitive and effective tool to identify and quantify potential biomarkers in repeated dose toxicity studies using primary cell culture systems. In this respect, 8-plex isobaric tag for relative and absolute quantification labeling is the method of choice for relative quantification. After cell lysis and tryptic protein digestion, an individual isobaric tag is added to the amine groups of arginine and lysine. Then, up to eight differentially labeled samples are mixed and analyzed together in a mass spectrometry experiment. During peptide fragmentation in the mass spectrometer, the individual tag intensity of each identified peptide could be detected, reflecting the peptide intensities in the eight samples. The identified peptides are matched to their specific protein using specific search engines and finally to eight individual relative protein quantities. The two-dimensional fractionation of complex peptide mixtures minimizes the possibility of co-fragmentation of peptides from different origin in the mass spectrometer, which leads to a higher number of peptide search matches and therefore to better identification and quantification results.

Proteomic analysis of nasal epithelial cells from cystic fibrosis patients.

The pathophysiology of cystic fibrosis (CF) lung disease remains incompletely understood. New explanations for the pathogenesis of CF lung disease may be discovered by studying the patterns of protein expression in cultured human nasal epithelial cells (HNEC). To that aim, we compared the level of protein expressions in primary cultures of HNEC from nasal polyps secondary to CF (CFNP, n = 4), primary nasal polyps (NP, n = 8) and control mucosa (CTRL, n = 4) using isobaric tag for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography (LC)-MS-MS. The analysis of the data revealed 42 deregulated protein expressions in CFNP compared to NP and CTRL, suggesting that these alterations are related to CF. Overall, AmiGo analysis highlighted six major pathways important for cell functions that seem to be impaired: metabolism, G protein process, inflammation and oxidative stress response, protein folding, proteolysis and structural proteins. Among them, glucose and fatty acid metabolic pathways could be impaired in CF with nine deregulated proteins. Our proteomic study provides a reproducible set of differentially expressed proteins in airway epithelial cells from CF patients and reveals many novel deregulated proteins that could lead to further studies aiming to clarify the involvement of such proteins in CF pathophysiology.

Quantitative proteomic analysis of serum proteins from oral cancer patients: comparison of two analytical methods.

Serum proteomic analysis can be a valuable approach for the discovery of protein biomarkers for early detection or monitoring of a disease. In this study, two analytical methods were compared for quantification of serum proteins in patients with oral cancer. In the first approach, we quantified serum proteins between oral squamous cell carcinoma (OSCC) and healthy control subjects by performing in-solution digestion of serum proteins, isobaric tags for relative and absolute quantification (iTRAQ) labeling of the resulting peptides, strong cation exchange (SCX) fractionation of labeled peptides and finally capillary liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis of the peptides. In the second approach, we first separated serum proteins with SDS-PAGE. The gel-separated proteins were then digested with trypsin and the resulting peptides were labeled with iTRAQ and analyzed with LC-MS/MS for protein quantification. A total of 319 serum proteins were quantified with the first proteomic approach whereas a total of 281 proteins were quantified by the second proteomic approach. Most of the proteins were identified and quantified by both approaches, suggesting that these methods are similarly effective for serum proteome analysis. This study provides compelling evidence that quantitative serum proteomic analysis of OSCC is a valuable approach for identifying differentially expressed proteins in cancer patients’ circulation systems that may be used as potential biomarkers for disease detection. Further validation in large oral cancer patient populations may lead to a simple and low invasive clinical tool for OSCC diagnosis or monitoring.

Proteomic alteration of PK-15 cells after infection by porcine circovirus type 2.

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of post-weaning multisystemic wasting syndrome, which has spread worldwide. To discover cellular protein responses of PK-15 cells to PCV2 infection, two-dimensional liquid chromatography–tandem mass spectrometry (MS) coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the proteins that were differentially expressed in PK-15 from the PCV2-infected group compared to the uninfected control group. A total of 196 cellular proteins in PK-15 that were significantly altered at different time periods post-infection were identified. These differentially expressed proteins were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc. and their interactions. Moreover, some of these proteins were further confirmed by Western blot. The high number of differentially expressed proteins identified should be very useful in elucidating the mechanism of replication and pathogenesis of PCV2 in the future.

Plasma proteomic changes during hypothermic and normothermic cardiopulmonary bypass in aortic surgeries.

Deep hypothermic circulatory arrest (DHCA) is a protective method against brain ischemia in aortic surgery. However, the possible effects of DHCA on the plasma proteins remain to be determined. In the present study, we used novel high-throughput technology to compare the plasma proteomes during DHCA (22°C) with selective cerebral perfusion (SCP, n=7) to those during normothermic cardiopulmonary bypass (CPB, n=7). Three plasma samples per patient were obtained during CPB: T1, prior to cooling; T2, during hypothermia; T3, after rewarming for the DHCA group and three corresponding points for the normothermic group. A proteomic analysis was performed using isobaric tag for relative and absolute quantification (iTRAQ) labeling tandem mass spectrometry to assess quantitative protein changes. In total, the analysis identified 262 proteins. The bioinformatics analysis revealed a significant upregulation of complement activation at T2 in normothermic CPB, which was suppressed in DHCA. These findings were confirmed by the changes of the terminal complement complex (SC5b-9) levels. At T3, however, the level of SC5b-9 showed a greater increase in DHCA compared to normothermic CPB, while 48 proteins were significantly downregulated in DHCA. The results demonstrated that DHCA and rewarming potentially exert a significant effect on the plasma proteome in patients undergoing aortic surgery.

Proteomic Analysis of Saliva from Patients with Oral Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD) is an immune-mediated disorder and is the major long-term complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The oral mucosa, including the salivary glands, is affected in the majority of patients with cGVHD; however, at present there is only a limited understanding of disease pathobiology. In this study, we performed a quantitative proteomic analysis of saliva pooled from patients with and without oral cGVHD—cGVHD(+) and cGVHD(−), respectively—using isobaric tags for relative and absolute quantification labeling, followed by tandem mass spectrometry. Among 249 salivary proteins identified by tandem mass spectrometry, 82 exhibited altered expression in the oral cGVHD(+) group compared with the cGVHD(−) group. Many of the identified proteins function in innate or acquired immunity, or are associated with tissue maintenance functions, such as proteolysis or the cytoskeleton. Using ELISA immunoassays, we further confirmed that 2 of these proteins, IL-1 receptor antagonist and cystatin B, showed decreased expression in patients with active oral cGVHD (P < .003). Receiver operating curve characteristic analysis revealed that these 2 markers were able to distinguish oral cGVHD with a sensitivity of 85% and specificity of 60%, and showed slightly better discrimination in newly diagnosed patients evaluated within 12 months of allo-HSCT (sensitivity, 92%; specificity 73%). In addition to identifying novel potential salivary cGVHD biomarkers, our study demonstrates that there is coordinated regulation of protein families involved in inflammation, antimicrobial defense, and tissue protection in oral cGVHD that also may reflect changes in salivary gland function and damage to the oral mucosa.

Quantitative iTRAQ LC-MS/MS Proteomics Reveals Transcription Factor Crosstalk and Regulatory Networks in Hypopharyngeal Squamous Cell Carcinoma.

To date, no effective therapeutic treatments have been developed for hypopharyngeal squamous cell carcinoma (HPSCC), a disease that has a five-year survival rate of approximately 31% because of its late diagnosis and aggressive nature. Despite recent improvements in diagnostic methods, there are no effective measures to prevent or detect HPSCC in an early stage. The goal of the current study was to identify molecular biomarkers and networks that can facilitate the speedy identification of HPSCC patients who could benefit from individualized treatment. Isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed with two-dimensional liquid chromatography-tandem mass spectrometry to identify quantitatively the differentially expressed proteins among three types of HPSCC disease stages. The iTRAQ results were evaluated by literature searches and western blot analysis. For example, FUBP1, one of 412 proteins with significantly altered expression profiles, was confirmed to have elevated expression in fresh HPSCC tissues. Integrin-mediated cell matrix adhesion and actin filament-inducing cytoskeleton remodeling were the cellular events that were the most relevant to HPSCC tumorigenesis and the metastatic process. The construction of transcriptional regulation networks led to the identification of key transcriptional regulators of tumor development and lymph node metastasis of HPSCC, including Sp1, c-Myc and p53. Additionally, our study indicated that the interactions among Sp1, c-Myc and p53 may play vital roles in the carcinogenesis and metastasis of HPSCC.

Identification of differentially expressed proteins in fresh and frozen–thawed boar spermatozoa by iTRAQ-coupled 2D LC–MS/MS

Cryodamage is a major problem in semen cryopreservation, causing changes in the levels of proteins that influence the function and motility of spermatozoa. In this study, protein samples prepared from fresh and frozen-thawed boar spermatozoa were compared using the isobaric tags for relative and absolute quantification (iTRAQ) labeling technique coupled to 2D LC-MS/MS analysis. A total of 41 differentially expressed proteins were identified and quantified, including 35 proteins that were present at higher levels and six proteins that were present at lower levels in frozen-thawed spermatozoa by at least a mean of 1.79-fold (P<0.05). On classifying into ten distinct categories using bioinformatic analysis, most of the 41 differentially expressed proteins were found to be closely relevant to sperm premature capacitation, adhesions, energy supply, and sperm-oocyte binding and fusion. The expression of four of these proteins, SOD1, TPI1, ODF2, and AKAP3, was verified by western blot analysis. We propose that alterations in these identified proteins affect the quality of cryopreserved semen and ultimately lower its fertilizing capacity. This is the first study to compare protein levels in fresh and frozen-thawed spermatozoa using the iTRAQ technology. Our preliminary results provide an overview of the molecular mechanisms of cryodamage in frozen-thawed spermatozoa and theoretical guidance to improve the cryopreservation of boar semen.

ITRAQ Analysis with Paul Ion Trap–Obstacle Solved

Mass spectrometers equipped with ion trap analyzers have been significantly improved due to their high performance and wide application area accompanying the low costs of purchase. Despite several advantages, such as reasonable resolution at low cost, high sensitivity, and capability for multistage analysis, ion traps have an important drawback: low mass cutoff during tandem mass spectrometry analysis MS(n). Although the low mass cutoff associated with the ion trap does not seriously obstruct peptide identification, it may cause a serious problem in identification of small molecules (posttranslational modifications, e.g., glycan structures) and quantification of peptides with multiplexed isobaric tag reagents. The presented approach offers the possibility to use isobaric tags for relative and absolute quantification labeling (iTRAQ) for quantitative, proteomic analysis using typical, widely available ion trap devices and manufacturer's software. We have performed series of analyses of standard protein labeled with isobaric tags in various concentration ratios to prove quantitative capabilities of this approach.

Proteome-wide Dysregulation by Glucose-6-phosphate Dehydrogenase (G6PD) Reveals a Novel Protective Role for G6PD in Aflatoxin B1-Mediated Cytotoxicity

Glucose-6-phosphate dehydrogenase (G6PD) is pivotal to reduced nicotinamide adenine dinucleotide phosphate (NADPH) production and cellular redox balance. Cells with G6PD deficiency are susceptible to oxidant-induced death at high oxidative stress. However, it remains unclear what precise biological processes are affected by G6PD deficiency due to altered cellular redox homeostasis, particularly at low oxidative stress. To further explore the biological role of G6PD, we generated G6PD-knockdown cell clones using lung cancer line A549. We identified proteins differentially expressed in the knockdown clones without the addition of exogenous oxidant by means of isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-mass spectrometry (LC-MS/MS). We validated a panel of proteins that showed altered expression in G6PD-knockdown clones and were involved in metabolism of xenobiotic and glutathione (GSH) as well as energy metabolism. To determine the physiological relevancy of our findings, we investigated the functional consequence of G6PD depletion in cells treated with a prevalent xenobiotic, aflatoxin B₁(AFB₁). We found a protective role of G6PD in AFB₁-induced cytotoxicity, possibly via providing NADPH for NADPH oxidase to induce epoxide hydrolase 1 (EPHX1), a xenobiotic-metabolizing enzyme. Collectively, our findings reveal for the first time a proteome-wide dysregulation by G6PD depletion under the condition without exogenous oxidant challenge, and we suggest a novel association of G6PD activity with AFB₁-related xenobiotic metabolism.

Two-dimensional liquid chromatography–tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling approach revealed first proteome profiles of pulmonary alveolar macrophages infected with porcine circovirus type 2

Porcine circovirus type 2 (PCV2) has been identified as the essential causal agent of postweaning multisystemic wasting syndrome, which has spread worldwide. Monocyte/macrophage lineage cells are the major target cells of PCV2. To discover cellular protein responses of pulmonary alveolar macrophages (PAMs) to PCV2 infection, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the proteins that were differentially expressed in PAMs from the PCV2-infected group compared to the uninfected control group. A total of 145 cellular proteins in PAMs that were significantly altered at different time periods post-infection were identified. These differentially expressed proteins were related to the biological processes of binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. The high number of differentially expressed proteins identified should be very useful to elucidate the mechanism of replication and pathogenesis of PCV2 in the future.

DNA Ends Alter the Molecular Composition and Localization of Ku Multicomponent Complexes

The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

Two-Dimensional Liquid Chromatography–Tandem Mass Spectrometry Coupled with Isobaric Tags for Relative and Absolute Quantification (iTRAQ) Labeling Approach Revealed First Proteome Profiles of Pulmonary Alveolar Macrophages Infected with Porcine Reproductive and Respiratory Syndrome Virus

Porcine reproductive and respiratory syndrome (PRRS) has devastated the pig industry worldwide for almost 25 years, and its virus (PRRSV) preferentially infects and replicates in pulmonary alveolar macrophages (PAMs). To discover cellular protein responses in PRRSV-infected PAMs, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins between the PRRSV-infected groups and the controls. A total of 160 cellular proteins in PAMs that were significantly altered post-infection were identified. These differentially expressed proteins are related to the biological processes of virus binding, cell structure, signal transduction, cell adhesion, etc., and their interactions. This is the first report that analyzed the cellular protein profile of PRRSV-infected PAMs using iTRAQ technology, and this data provides important information to help understand the host response to PRRSV and to define the cellular requirements for the underlying mechanism of PRRSV replication and pathogenesis.

Quantitative proteomic analysis of serum proteins in patients with Parkinson’s disease using an isobaric tag for relative and absolute quantification labeling, two-dimensional liquid chromatography, and tandem mass spectrometry

Parkinson's disease (PD) is a common disease which occurs in aged people with chronic, progressive degenerative character of the central nervous system. Until now there is no effective treatment method in PD patients before they show obvious symptoms for prevention and early diagnosis. In order to find out early disease specific biomarkers, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the different disease progress types of PD. 26 proteins were differentially expressed in a total of 258 identified proteins by proteomic techniques. The expression level of eight proteins which included sero-transferrin and clusterin increased. The expression level of eighteen proteins which include complement component 4B, apolipoprotein A-I, α-2-antiplasmin and coagulation factor V decreased. Those proteins may be associated with oxidative stress, mitochondrial dysfunction, abnormal protein aggregation and inflammation. In this study, the expression level of apolipoprotein A-I decreased, particularly in the early stage of PD patients. This protein regulated not only the lipid metabolism in the central nervous system, but also influenced the deposition process of proteins which are involved in neural degenerative diseases, such as the pathogenesis of PD.

Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns

The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC–MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called “sponge effect” was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC–MALDI-TOF/TOF-MS analysis was less than 17.5%.

A Gel-Free Quantitative Proteomics Analysis of Factors Released From Hypoxic-Conditioned Placentae

Characterizing the protein factors released from placentae during pathogenesis remains a key objective toward understanding preeclampsia and related pregnancy disorders. Gel-free proteomics technologies applied to placental explant-conditioned media offers the potential of identifying these factors. Relative quantification mass spectrometry using isobaric tagging for relative and absolute quantification (iTRAQ) labeling was employed to compare the ''secretome'' between healthy term placental tissue cultured under both normoxic and hypoxic oxygen tensions. Of the 499 proteins identified, 45 were differentially expressed (P < .01 level), including interleukin 8 (IL-8) which was significantly upregulated under hypoxia. Global protein level changes are suggestive of decreased extracellular matrix remodeling under the same conditions. A significant enrichment of soluble liberated placental factors is achieved using this model system. Identifying these changes resulting from hypoxic conditioning is hypothesis generating and may provide new mechanistic insights into preeclampsia.

Quantitative Proteomic Analysis of Formalin-fixed and Paraffin-embedded Nasopharyngeal Carcinoma Using iTRAQ Labeling, Two-dimensional Liquid Chromatography, and Tandem Mass Spectrometry

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens represent a potentially valuable resource for protein biomarker investigations. In this study, proteins were extracted by a heat-induced antigen retrieval technique combined with a retrieval solution containing 2% SDS from FFPE tissues of normal nasopharyngeal epithelial tissues (NNET) and three histological types of nasopharyngeal carcinoma (NPC) with diverse differentiation degrees. Then two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the types of NPC FFPE tissues. Our study resulted in the identification of 730 unique proteins, the distributions of subcellular localizations and molecular functions of which were similar to those of the proteomic database of human NPC and NNET that we had set up based on the frozen tissues. Additionally, the relative expression levels of cathepsin D, keratin8, SFN, and stathmin1 identified and quantified in this report were consistent with the immunohistochemistry results acquired in our previous study. In conclusion, we have developed an effective approach to identifying protein changes in FFPE NPC tissues utilizing iTRAQ technology in conjunction with an economical and easily accessible sample preparation method.

Quantitative Plasma Proteomic Profiling Identifies the Vitamin E Binding Protein Afamin as a Potential Pathogenic Factor in SIV Induced CNS Disease

Investigating, predicting, diagnosing, and treating HIV-1-associated neurocognitive disorder (HAND) has been hindered by the lack of disease-related molecular markers. In this study, plasma from rhesus monkeys (n = 6), before and after infection with simian immunodeficiency virus (SIV), was profiled to obtain differential fingerprints in protein expression during SIV-induced central nervous system (CNS) disease. A quantitative proteomic analysis was performed by means of isobaric tag for relative and absolute quantification (iTRAQ) labeling, using multidimensional liquid chromatography tandem mass spectrometry (LC-MS/MS) run on a linear ion trap mass spectrometer in an integrated mode comprising pulsed-Q-dissociation (PQD) and CID. Among a panel of proteins showing differential expression following SIV infection, we identified afamin, a member of the albumin superfamily, to be significantly down regulated after infection. Validation by Western blot confirmed this observation and, given its potential implication in neuroprotection by transport of alpha-tocopherol (alphaTocH), provides new avenues into further understanding HIV induced CNS disease. iTRAQ-based LC-MS/MS provides a valuable platform for plasma protein profiling and has important implications in identifying molecular markers relevant for the pathogenesis of neurodegenerative diseases. Using such an approach, we show its successful application in identifying differential fingerprints in SIV/HIV induced CNS disease.

Quantitative Proteomics Identifies Surfactant-Resistant α-Synuclein in Cerebral Cortex of Parkinsonism-Dementia Complex of Guam but Not Alzheimer's Disease or Progressive Supranuclear Palsy

Parkinsonism-dementia complex (PDC) remains a significant health burden to the Chamorro population. We tested the hypothesis that quantitative proteomics might provide fresh insight into this enigmatic illness by analyzing proteins resistant to surfactant extraction from patients with Alzheimer's disease (AD) or PDC and their matched controls using isobaric tags for relative and absolute quantification. In addition to the expected increase in abnormal frontal cortical Abeta peptides, tau, ubiquitin, and apolipoprotein E in AD, and tau in PDC, we identified alpha-synuclein (SNCA) as a major abnormal protein in PDC but not AD. We confirmed our isobaric tags for relative and absolute quantification findings by enzyme-linked immunosorbent assay in frontal and temporal cortices. We extended our assays to include a limited number of cases of progressive supranuclear palsy (PSP) and dementia with Lewy bodies; we observed increased abnormal tau but not SNCA in PSP, and abnormal SNCA in dementia with Lewy bodies that was quantitatively similar to PDC. Finally, soluble Abeta oligomers were selectively increased in AD but not PDC or PSP. These results show that frontal and temporal cortex in PDC is distinguished from AD and PSP by its accumulation of abnormal SNCA and suggest that PDC be considered a synucleinopathy as well as a tauopathy.

Quantitative Comparison of IMAC and TiO 2 Surfaces Used in the Study of Regulated, Dynamic Protein Phosphorylation

Protein phosphorylation regulates many aspects of cellular function, including cell proliferation, migration, and signal transduction. An efficient strategy to isolate phosphopeptides from a pool of unphosphorylated peptides is essential to global characterization using mass spectrometry. We describe an approach employing isotope tagging reagents for relative and absolute quantification (iTRAQ) labeling to compare quantitatively commercial and prototypal immobilized metal affinity chelate (IMAC) and metal oxide resins. Results indicate a prototype iron chelate resin coupled to magnetic beads outperforms either the Ga(3+)-coupled analog, Fe(3+), or Ga(3+)-loaded, iminodiacetic acid (IDA)-coated magnetic particles, Ga(3+)-loaded Captivate beads, Fe(3+)-loaded Poros 20MC, or zirconium-coated ProteoExtract magnetic beads. For example, compared with Poros 20MC, the magnetic metal chelate (MMC) studied here improved phosphopeptide recovery by 20% and exhibited 60% less contamination from unphosphorylated peptides. With respect to efficiency and contamination, MMC performed as well as prototypal magnetic metal oxide-coated (TiO(2)) beads (MMO) or TiO(2) chromatographic spheres, even if the latter were used with 2,5-dihydroxybenzoic acid (DHB) procedures. Thus far, the sensitivity of the new prototypes reaches 50 fmol, which is comparable to TiO(2) spheres. In an exploration of natural proteomes, tryptic (phospho)peptides captured from stable isotopic labeling with amino acids in cell culture (SILAC)-labeled immunocomplexes following EGF-treatment of 5 x 10(7) HeLa cells were sufficient to quantify stimulated response of over 60 proteins and identify 20 specific phosphorylation sites.