RESUMEN
This work demonstrates the utility of high-throughput nanoLC-MS and label-free quantification (LFQ) for sample-limited bottom-up proteomics analysis, including single-cell proteomics (SCP). Conditions were optimized on a 50 µm internal diameter (I.D.) column operated at 100 nL/min in the direct injection workflow to balance method sensitivity and sample throughput from 24 to 72 samples/day. Multiple data acquisition strategies were also evaluated for proteome coverage, including data-dependent acquisition (DDA), wide-window acquisition (WWA), and wide-window data-independent acquisition (WW-DIA). Analyzing 250 pg HeLa digest with a 10-min LC gradient (72 samples/day) provided >900, >1,800, and >3,000 protein group identifications for DDA, WWA, and WW-DIA, respectively. Total method cycle time was further reduced from 20 to 14.4 min (100 samples/day) by employing a trap-and-elute workflow, enabling 70% mass spectrometer utilization. The method was applied to library-free DIA analysis of single-cell samples, yielding >1,700 protein groups identified. In conclusion, this study provides a high-sensitivity, high-throughput nanoLC-MS configuration for sample-limited proteomics.
Asunto(s)
Cromatografía Líquida con Espectrometría de Masas , Proteómica , Humanos , Proteómica/métodos , Cromatografía Liquida , Espectrometría de Masas en Tándem/métodos , Proteoma/análisisRESUMEN
This study demonstrates how the latest ultrahigh-performance liquid chromatography (UHPLC) technology can be combined with high-resolution accurate-mass (HRAM) mass spectrometry (MS) and long columns packed with fully porous particles to improve bottom-up proteomics analysis with nanoflow liquid chromatography-mass spectrometry (nanoLC-MS) methods. The increased back pressures from the UHPLC system enabled the use of 75 µm I.D. × 75 cm columns packed with 2 µm particles at a typical 300 nL/min flow rate as well as elevated and reduced flow rates. The constant pressure pump operation at 1500 bar reduced sample loading and column washing/equilibration stages and overall overhead time, which maximizes MS utilization time. The versatility of flow rate optimization to balance the sensitivity, throughput with sample loading amount, and capability of using longer gradients contributes to a greater number of peptide and protein identifications for single-shot bottom-up proteomics experiments. The routine proteome profiling and precise quantification of >7000 proteins with single-shot nanoLC-MS analysis open possibilities for large-scale discovery studies with a deep dive into the protein level alterations. Data are available via ProteomeXchange with identifier PXD035665.
Asunto(s)
Péptidos , Proteoma , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida , Espectrometría de Masas , Péptidos/análisis , Porosidad , Proteoma/análisisRESUMEN
A current trend in proteomics is to acquire data in a "single-shot" by LC-MS/MS because it simplifies workflows and promises better throughput and quantitative accuracy than schemes that involve extensive sample fractionation. However, single-shot approaches can suffer from limited proteome coverage when performed by data dependent acquisition (ssDDA) on nanoflow LC systems. For applications where sample quantities are not scarce, this study shows that high proteome coverage can be obtained using a microflow LC-MS/MS system operating a 1 mm i.d. × 150 mm column, at a flow-rate of 50 µL/min and coupled to an Orbitrap HF-X mass spectrometer. The results demonstrate the identification of â¼9â¯000 proteins from 50 µg of protein digest from Arabidopsis roots, 7â¯500 from mouse thymus, and 7â¯300 from human breast cancer cells in 3 h of analysis time in a single run. The dynamic range of protein quantification measured by the iBAQ approach spanned 5 orders of magnitude and replicate analysis showed that the median coefficient of variation was below 20%. Together, this study shows that ssDDA by µLC-MS/MS is a robust method for comprehensive and large-scale proteome analysis and which may be further extended to more rapid chromatography and data independent acquisition approaches in the future.Ì.
Asunto(s)
Cromatografía Liquida , Proteómica , Espectrometría de Masas en Tándem , Animales , Arabidopsis , Línea Celular , Humanos , Ratones , ProteomaRESUMEN
Although current LC-MS technology permits scientists to efficiently screen clinical samples in translational research, e.g., steroids, biogenic amines, and even plasma or serum proteomes, in a daily routine, maintaining the balance between throughput and analytical depth is still a limiting factor. A typical approach to enhance the proteome depth is employing offline two-dimensional (2D) fractionation techniques before reversed-phase nanoLC-MS/MS analysis (1D-nanoLC-MS). These additional sample preparation steps usually require extensive sample manipulation, which could result in sample alteration and sample loss. Here, we present and compare 1D-nanoLC-MS with an automated online-2D high-pH RP × low pH RP separation method for deep proteome profiling using a nanoLC system coupled to a high-resolution accurate-mass mass spectrometer. The proof-of-principle study permitted the identification of ca. 500 proteins with â¼10,000 peptides in 15 enzymatically digested crude serum samples collected from healthy donors in 3 laboratories across Europe. The developed method identified 60% more peptides in comparison with conventional 1D nanoLC-MS/MS analysis with ca. 4 times lower throughput while retaining the quantitative information. Serum sample preparation related changes were revealed by applying unsupervised classification techniques and, therefore, must be taken into account while planning multicentric biomarker discovery and validation studies. Overall, this novel method reduces sample complexity and boosts the number of peptide and protein identifications without the need for extra sample handling procedures for samples equivalent to less than 1 µL of blood, which expands the space for potential biomarker discovery by looking deeper into the composition of biofluids.
Asunto(s)
Proteoma , Espectrometría de Masas en Tándem , Cromatografía Liquida , Proteómica , Manejo de EspecímenesRESUMEN
Microflow liquid chromatography tandem mass spectrometry (µLC-MS/MS) is becoming a viable alternative to nanoflow LC-MS/MS for the analysis of proteomes. We have recently demonstrated the potential of such a system operating with a 1 mm i.d. × 150 mm column and at a flow rate of 50 µL/min for high-throughput applications. On the basis of the analysis of â¼38â¯000 samples measured on two instruments over the past two years, we now show that the approach is extremely robust. Up to 1500 analyses were performed within one month, and >14â¯000 samples could be analyzed on a single column without loss of chromatographic performance. Samples included proteomes of cell lines, tissues, and human body fluids, which were analyzed with or without prior peptide fractionation or stable isotope labeling. We show that the µLC-MS/MS system is capable of measuring 2600 proteins from undepleted human plasma and â¼5000 proteins from crude human urine in 1 day, demonstrating its potential for in-depth as well as high-throughput clinical application.
Asunto(s)
Proteoma , Espectrometría de Masas en Tándem , Cromatografía Liquida , Humanos , Marcaje Isotópico , PéptidosRESUMEN
Most molecular cancer therapies act on protein targets but data on the proteome status of patients and cellular models for proteome-guided pre-clinical drug sensitivity studies are only beginning to emerge. Here, we profiled the proteomes of 65 colorectal cancer (CRC) cell lines to a depth of > 10,000 proteins using mass spectrometry. Integration with proteomes of 90 CRC patients and matched transcriptomics data defined integrated CRC subtypes, highlighting cell lines representative of each tumour subtype. Modelling the responses of 52 CRC cell lines to 577 drugs as a function of proteome profiles enabled predicting drug sensitivity for cell lines and patients. Among many novel associations, MERTK was identified as a predictive marker for resistance towards MEK1/2 inhibitors and immunohistochemistry of 1,074 CRC tumours confirmed MERTK as a prognostic survival marker. We provide the proteomic and pharmacological data as a resource to the community to, for example, facilitate the design of innovative prospective clinical trials.
Asunto(s)
Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica , Tirosina Quinasa c-Mer/genética , Biomarcadores de Tumor/antagonistas & inhibidores , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/mortalidad , Redes Reguladoras de Genes , Humanos , Inmunohistoquímica , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa 2/antagonistas & inhibidores , MAP Quinasa Quinasa 2/genética , MAP Quinasa Quinasa 2/metabolismo , Farmacogenética/métodos , Pronóstico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteómica/métodos , Transducción de Señal , Análisis de Supervivencia , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Tirosina Quinasa c-Mer/metabolismoRESUMEN
Offline two-dimensional chromatography is a common means to achieve deep proteome coverage. To reduce sample complexity and dynamic range and to utilize mass spectrometer (MS) time efficiently, high chromatographic resolution of and good orthogonality between the two dimensions are needed. Ion exchange and high pH reversed phase chromatography are often used for this purpose. However, the former requires desalting to be MS-compatible, and the latter requires fraction pooling to create orthogonality. Here, we report an alternative first-dimension separation technique using a commercial trimodal phase incorporating polar embedded reversed phase, weak anion exchange, and strong cation exchange material. The column is capable of retaining polar and nonpolar peptides alike without noticeable breakthrough. It allows separating ordinary and TMT-labeled peptides under mild acidic conditions using an acetonitrile gradient. The direct MS compatibility of solvents and good orthogonality to online coupled C18 columns enable a straightforward workflow without fraction pooling and desalting while showing comparable performance to the other techniques. The method scales from low to high microgram sample quantity and is amenable to full automation. To demonstrate practical utility, we analyzed the proteomes of 10 human pancreatic cancer cell lines to a depth of >8,700 quantified proteins.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Proteoma/análisis , Espectrometría de Masas en Tándem/métodos , Línea Celular Tumoral , Cromatografía por Intercambio Iónico , Cromatografía de Fase Inversa , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos/análisis , Péptidos/aislamiento & purificación , Proteoma/aislamiento & purificación , Reproducibilidad de los Resultados , Tiazoles/químicaRESUMEN
In this paper we describe a rapid, simple, and costeffective liquid chromatographytandem mass spectrometric (LCMSMS) method for simultaneous analysis of aflatoxin B1, B2, G1, and G2, ochratoxin A, and sterigmatocystin in 25 traditional Chinese medicines (TCMs). The method is based on single extraction with 84:16 (v/v) acetonitrilewater then analysis of the diluted crude extract without further clean-up. Chromatographic separation was achieved on a C18 column, with a mobile phase gradient prepared from aqueous 4 mmol L−1 ammonium acetate0.1 % formic acid and methanol. Quantification of the analytes was by selective reaction monitoring (SRM) on a triple-quadrupole mass spectrometer in positive-ionization mode. Special focus was on investigating and reducing matrix effects to improve accuracy. The established method was validated by determination of linearity (r>0.995), sensitivity (limits of quantification 1.625.0 ng L−1), apparent recovery (84.8110.6 %), extraction recovery (83.6106.1 %), and precision (relative standard deviation ≤9.9 %) for two representative TCMs, Semen Armeniacae Amarae and Radix Pseudostellariae. The applicability of the method to TCMs other than these was further investigated, and 23 other TCMs with acceptable matrix effects (80.2118.6 %) were screened. The validated method was finally used to assess mycotoxin contamination of 244 samples of 25 TCMs collected from local hospitals and TCM pharmacies. Aflatoxin B1 and ochratoxin A were detected in 5.3 % of the samples. Sterigmatocystin, the most prevalent mycotoxin contaminant, was present in 26.2 % of the samples tested; this has not been reported previously. The results of this work imply greater attention should be devoted to evaluation of the potential hazard caused by sterigmatocystin in TCMs.
Asunto(s)
Aflatoxina B1/análisis , Aflatoxinas/análisis , Cromatografía Liquida/métodos , Medicina Tradicional China , Ocratoxinas/análisis , Esterigmatocistina/análisis , Espectrometría de Masas en Tándem/métodos , Contaminación de Medicamentos , Hospitales , HumanosRESUMEN
As the dilution procedure was applied, a simple, rapid and cost-effective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of aflatoxin B1, B2, G1, and G2 was successfully by performed in a total 83 samples of 10 traditional Chinese medicines (TCMs), which were collected from 5 different hospital pharmacies and 5 different medical stores in Guangzhou city. Matrix effects of these 10 TCMs were ranged from 80.23% to 115.5% in low, intermediate and high concentration levels, indicating that the negative effect was overcome in this study. Meanwhile, the analysis method was proved to be stable and reliable during the whole analysis using Semen Armeniacae Amarum spiked 3 concentration levels of standard solution as quality control samples and the RSD < 6.6% was obtained. The contamination levels of 83 investigated samples were 13.89% and 17.02% in hospital pharmacies and medical stores, respectively. The result was presented to provide relevant reference and supplement to those researchers in TCMs analysis and screening.
Asunto(s)
Aflatoxinas/análisis , Cromatografía Líquida de Alta Presión/métodos , Contaminación de Medicamentos , Medicina Tradicional China , Espectrometría de Masas en Tándem/métodos , Aflatoxina B1/análisis , Control de CalidadRESUMEN
A simple, rapid and cost-effective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/ MS) method was established for simultaneous determination of aflatoxins (AFB1, AFB2, AFG1, AFG2) in Armeniacae Semen Amarum and the application was performance in 11 samples collected from different markets, medical stores and hospitals. The sample was extracted with 84% acetonitrile/water and 250 microL extraction was directly injected into a LC-MS/MS system without further purification procedure after being redissolved with methanol. The LC separation was performed on a C18 column with a linear gradient elution program of 4 mmol x L(-1) NH4 Ac-0.1% formic acid solution and menthol as the mobile phase. Selected reaction monitoring (SRM) was used for selective determination of the four aflatoxins on a triple quadruple mass spectrometer, which was operated in positive ionization modes. All the four aflatoxins showed a good linear relationship with r > 0.999 0, the average recoveries were between 87.88% and 102.9% and the matrix effect was ranged from 90.71% to 99.30% in low, intermediate and high levels. Furthermore, the higher recovery was obtained by the method reported in this study, comparing to the cleanup procedure with the Mycosep 226 purification column. Eleven samples collected were detected and the contamination levels of the AFB1 were between 1.590-2.340 microg x kg(-1) and the AF (B1 + B2 + G1 + G2) was ranged from 2.340 to 3.340 microg x kg(-1). In summary, the developed method was suitable to detect and screen AFB1, AFB2, AFG1, AFG2 in Armeniacae Semen Amarum.
Asunto(s)
Aflatoxinas/análisis , Cromatografía Líquida de Alta Presión/métodos , Contaminación de Medicamentos , Prunus/química , Semillas/química , Espectrometría de Masas en Tándem/métodos , Aflatoxinas/metabolismo , Hongos/metabolismo , Prunus/microbiología , Semillas/microbiologíaRESUMEN
OBJECTIVE: To evaluate fungal contamination on the surface of Chinese herbal medicines and explore an appropriate method for fast and efficient identification of contaminant fungi. METHOD: Chinese herbal medicines were first washed and the washing solution was plated onto potato dextrose agar (PDA) to obtain the pure isolates. For molecular identification, two new pairs of specific primers were designed according to ITS region of fungi genome sequences. The strains were identified through polymerase chain reaction (PCR) and sequence analysis. RESULT: Fifty fungal strains were obtained from the surface of 15 Chinese herbal medicines with the percent of contaminated samples of 93.3%. Twenty-seven strains among them were successfully identified. CONCLUSION: Fungal contamination on the surface of Chinese herbal medicines is quite common. Although different fungal species were isolated, the genus Aspergillus was the predominant. The primer pairs developed in this study are compatible and can be used to identify fungal species from the surface of Chinese herbal medicines.
Asunto(s)
Contaminación de Medicamentos , Medicamentos Herbarios Chinos , Hongos/aislamiento & purificación , Hongos/genética , Reacción en Cadena de la PolimerasaRESUMEN
Due to its important roles in oncogenic signaling, AKT has been subjected to extensive drug discovery efforts leading to small molecule inhibitors investigated in advanced clinical trials. To better understand how these drugs exert their therapeutic effects at the molecular level, we combined chemoproteomic target affinity profiling using kinobeads and phosphoproteomics to analyze the five clinical AKT inhibitors AZD5363 (Capivasertib), GSK2110183 (Afuresertib), GSK690693, Ipatasertib, and MK-2206 in BT-474 breast cancer cells. Kinobead profiling identified between four and 29 nM targets for these compounds and showed that AKT1 and AKT2 were the only common targets. Similarly, measuring the response of the phosphoproteome to the same inhibitors identified â¼1700 regulated phosphorylation sites, 276 of which were perturbed by all five compounds. This analysis expanded the known AKT signaling network by 119 phosphoproteins that may represent direct or indirect targets of AKT. Within this new network, 41 regulated phosphorylation sites harbor the AKT substrate motif, and recombinant kinase assays validated 16 as novel AKT substrates. These included CEP170 and FAM83H, suggesting a regulatory function of AKT in mitosis and cytoskeleton organization. In addition, a specific phosphorylation pattern on the ULK1-FIP200-ATG13-VAPB complex was found to determine the active state of ULK1, leading to elevated autophagy in response to AKT inhibition.
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Fosfoproteínas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Autofagia/efectos de los fármacos , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Nano-flow liquid chromatography tandem mass spectrometry (nano-flow LC-MS/MS) is the mainstay in proteome research because of its excellent sensitivity but often comes at the expense of robustness. Here we show that micro-flow LC-MS/MS using a 1 × 150 mm column shows excellent reproducibility of chromatographic retention time (<0.3% coefficient of variation, CV) and protein quantification (<7.5% CV) using data from >2000 samples of human cell lines, tissues and body fluids. Deep proteome analysis identifies >9000 proteins and >120,000 peptides in 16 h and sample multiplexing using tandem mass tags increases throughput to 11 proteomes in 16 h. The system identifies >30,000 phosphopeptides in 12 h and protein-protein or protein-drug interaction experiments can be analyzed in 20 min per sample. We show that the same column can be used to analyze >7500 samples without apparent loss of performance. This study demonstrates that micro-flow LC-MS/MS is suitable for a broad range of proteomic applications.
Asunto(s)
Cromatografía Liquida/métodos , Proteoma/análisis , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Línea Celular Tumoral , Células HeLa , Humanos , Péptidos/análisisRESUMEN
BACKGROUND: The dried parts of medicinal herbs are susceptible to the infection of fungi during pre- or post-harvest procedure. This study aimed to investigate the presence of fungi and their metabolites mycotoxins on the surface of medicinal herbs collected from China. METHODS: Forty-five retail samples of 15 different medicinal herbs were collected from 3 different regions in China. Then the potential fungi were immediately washed off from the surface of each sample with 0.1% Tween-20 followed by incubation of the rinse on petri-dish with potato dextrose agar containing chloramphenicol at 28 °C. The obtained fungi were isolated as single colonies and then characterized by morphology and molecular identification using internal transcribed spacer (ITS) sequencing with extracted DNA. Meanwhile, the mycotoxin-producing potential of the isolates was studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 126 fungi were identified from the surface of samples by morphology and ITS sequencing, with Aspergillus and Penicillium genera as the predominant contaminants. The mycotoxin-producing potential analysis showed that 6 of 8 A. versicolor isolates could produce sterigmatocystin. All 3 A. aculeatus isolates produced ochratoxin A, but only 1 of 3 A. flavus strains produced aflatoxins B1 and B2 without G1 and G2. Although the sample contamination ratios were high (≥95.6%), there was no significant difference (χ2 = 1.05, P = 1.0) among the samples from 3 regions, which demonstrates the prevalent fungal contamination in the herbal medicines. CONCLUSION: The prevalent contamination phenomenon of fungi and high potential risk of sterigmatocystin and ochratoxin A were observed in 45 medicinal herbs collected from China.