An off-line high pH reversed-phase fractionation and nano-liquid chromatography–mass spectrometry method for global proteomic profiling of cell lines

Wang, Hang; Sun, Shumin; Zhang, Yi; Chen, Si; Liu, Ping; Liu, Bin

doi:10.1016/j.jchromb.2014.10.031

Cited by 40 publications

(32 citation statements)

References 39 publications

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“…This “concatenation” tends to uniformly fill the gradients, leading to deeper proteomes independent of the nature of the sample, while maintaining throughput (20–22). This two-dimensional separation technique, combining high pH fractionation with concatenation, compares favorably with other approaches and is increasingly being applied by the proteomics community (20, 23–27). …”

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confidence: 99%

Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics

Kulak

Geyer

Mann

2017

Molecular & Cellular Proteomics

175

205

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Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven to be particularly powerful. This first dimension is typically performed with milliliter/min flow and relatively large column inner diameters, which allow efficient pre-fractionation but typically require peptide amounts in the milligram range. Here, we describe a novel approach termed “spider fractionator” in which the post-column flow of a nanobore chromatography system enters an eight-port flow-selector rotor valve. The valve switches the flow into different flow channels at constant time intervals, such as every 90 s. Each flow channel collects the fractions into autosampler vials of the LC-MS/MS system. Employing a freely configurable collection mechanism, samples are concatenated in a loss-less manner into 2–96 fractions, with efficient peak separation. The combination of eight fractions with 100 min gradients yields very deep coverage at reasonable measurement time, and other parameters can be chosen for even more rapid or for extremely deep measurements. We demonstrate excellent sensitivity by decreasing sample amounts from 100 μg into the sub-microgram range, without losses attributable to the spider fractionator and while quantifying close to 10,000 proteins. Finally, we apply the system to the rapid automated and in-depth characterization of 12 different human cell lines to a median depth of 11,472 different proteins, which revealed differences recapitulating their developmental origin and differentiation status. The fractionation technology described here is flexible, easy to use, and facilitates comprehensive proteome characterization with minimal sample requirements.

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confidence: 99%

Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics

Kulak

Geyer

Mann

2017

Molecular & Cellular Proteomics

175

205

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“…Gradient (300 nL/min) ACN/water/HFo Sproß et al (2013) et al, 2012; Shi et al, 2012;Hua et al, 2012;Su et al, 2015;Parker et al, 2013;Wang et al, 2015) or 2D polyacrylamide gel electrophoresis (PAGE) followed by in-gel digestion (Rogeberg et al, 2013;. Because this type of enzymatic digestion may require very long reaction times, a recent alternative is the employment of in-column immobilized enzymatic reactors for rapid digestion (seconds to minutes), coupled with a 2D analytical system (Xia et al, 2012;Sproß et al, 2013).…”

Section: Ms (Q-tof and Orbitrap)mentioning

confidence: 99%

“…9.4 shows a scheme of the procedure. In another example, Wang et al (2015) analyzed the fractions obtained from a conventional C 18 -silica column with high-pH conditions, in a nano-LC system with a C 18 -silica column.…”

Section: Ms (Q-tof and Orbitrap)mentioning

confidence: 99%

Theory and Practice of UHPLC and UHPLC–MS

Guillarme

Veuthey

2017

Handbook of Advanced Chromatography /Mass Spectrometry Techniques

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“…High-pH reversed-phase liquid chromatography (RPLC) provides better performance than the conventional strong cation-exchange chromatography. Combined with the second dimension of low-pH RPLC, the system improves both analytical dynamic range and protein coverage, yielding impressive results in whole proteome and phosphoproteome analyses (Batth et al, 2014; Song et al, 2010; Wang et al, 2015; Yang et al, 2012). For analyzing protein posttranslational modifications, affinity enrichment is critical as modified proteins are normally present in low stoichiometry.…”

Section: Introductionmentioning

confidence: 99%

“…Phosphopeptide enrichment by titanium dioxide (TiO 2 ) has been demonstrated to be a selective, efficient, and reproducible method, and is widely used (Beltran and Cutillas, 2012; Tan et al, 2015; Thingholm and Larsen, 2016). Other technical advances include small C18 particles (1.9 μm) and extended long column (~1 meter) (Wang et al, 2015). Other notable improvements are new versions of mass spectrometers with rapid scan rates, high sensitivity and high resolution (Hebert et al, 2014), and revised bioinformatics pipelines for MS data mining (Wang et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry

Bai

Tan

Pagala

et al. 2017

Methods in Enzymology

100

107

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Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. The major steps include protein extraction and digestion, TMT labeling, two dimensional liquid chromatography, TiO2-mediated phosphopeptide enrichment, high resolution mass spectrometry, and computational data processing. This protocol routinely leads to confident quantification of more than 10,000 proteins and approximately 30,000 phosphosites in mammalian samples. Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens.

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An off-line high pH reversed-phase fractionation and nano-liquid chromatography–mass spectrometry method for global proteomic profiling of cell lines

Cited by 40 publications

References 39 publications

Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics

Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics

Theory and Practice of UHPLC and UHPLC–MS

Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling, Extensive Liquid Chromatography, and Mass Spectrometry

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