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

View Article: PubMed Central - PubMed

ABSTRACT

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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Related in: MedlinePlus

Spider fractionation principle and practical implementation.A, switch mechanism of the rotor valve, illustrating how the flow from the first dimension separation is divided to eight output lines. B, schematic of the implementation of the spider fractionator. The first dimension separation is realized as a 250 μm inner diameter column, connected upstream through a zero dead volume connector to a nano-HPLC pump (an ultra high pressure unit is depicted but not required). The zoom-in is a cut-away symbolizing different peptide bands being separated in the column by different colors. Downstream, the column is connected to the rotor valve from A. The output lines feed into tubes that are filled in turn, according to the concatenation scheme. The spider-like appearance of the output lines give the name to the device. The arrows indicate that the output lines can be moved to a new set of tubes for a new separation process. After separation, the tubes are inserted into the autosampler of an UHPLC for LC-MS/MS analysis of the fractions. C, photo of the prototype spider fractionator used in this work.
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Figure 1: Spider fractionation principle and practical implementation.A, switch mechanism of the rotor valve, illustrating how the flow from the first dimension separation is divided to eight output lines. B, schematic of the implementation of the spider fractionator. The first dimension separation is realized as a 250 μm inner diameter column, connected upstream through a zero dead volume connector to a nano-HPLC pump (an ultra high pressure unit is depicted but not required). The zoom-in is a cut-away symbolizing different peptide bands being separated in the column by different colors. Downstream, the column is connected to the rotor valve from A. The output lines feed into tubes that are filled in turn, according to the concatenation scheme. The spider-like appearance of the output lines give the name to the device. The arrows indicate that the output lines can be moved to a new set of tubes for a new separation process. After separation, the tubes are inserted into the autosampler of an UHPLC for LC-MS/MS analysis of the fractions. C, photo of the prototype spider fractionator used in this work.

Mentions: The principle of the fractionator is depicted in Fig. 1. The post-column flow from the first dimension separation enters the input port of an eight-port flow-selector rotor valve. At pre-determined time intervals, the valve switches to a new output port. Each of the outputs is connected via a narrow bore capillary to different output lines in a sample collection device, distributing the sample flow into consecutive tubes for the pooled fractions. Once one cycle has been completed, the valves switches back to the first output port and the next fluid volume is added to the already collected first fraction. In this way, the device automatically concatenates and pools the samples, without requiring different collection tubes or the combination of separately collected effluent volumes. Therefore, the volumes are not constrained to a minimum size, which would otherwise be necessary to handle them in separate tubes. We routinely employ a 250 μm inner diameter column in the first dimension at 2 μl/min and switch the valve every 90 s, thus concatenation volumes are only 3 μl. The system is fully programmable, allowing collection not only into multiples of the eight output channels (A–H) but also into as few as two or as many fractions as there are collection tubes in the device (96 in our setup). Furthermore, an arbitrary number of samples can be fractionated, and the rotor valve shifts can be defined by the user. For example, 12 samples could be scheduled for concatenation into eight fractions each in a total of 24 h using 80 min gradients.


Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics *
Spider fractionation principle and practical implementation.A, switch mechanism of the rotor valve, illustrating how the flow from the first dimension separation is divided to eight output lines. B, schematic of the implementation of the spider fractionator. The first dimension separation is realized as a 250 μm inner diameter column, connected upstream through a zero dead volume connector to a nano-HPLC pump (an ultra high pressure unit is depicted but not required). The zoom-in is a cut-away symbolizing different peptide bands being separated in the column by different colors. Downstream, the column is connected to the rotor valve from A. The output lines feed into tubes that are filled in turn, according to the concatenation scheme. The spider-like appearance of the output lines give the name to the device. The arrows indicate that the output lines can be moved to a new set of tubes for a new separation process. After separation, the tubes are inserted into the autosampler of an UHPLC for LC-MS/MS analysis of the fractions. C, photo of the prototype spider fractionator used in this work.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5383787&req=5

Figure 1: Spider fractionation principle and practical implementation.A, switch mechanism of the rotor valve, illustrating how the flow from the first dimension separation is divided to eight output lines. B, schematic of the implementation of the spider fractionator. The first dimension separation is realized as a 250 μm inner diameter column, connected upstream through a zero dead volume connector to a nano-HPLC pump (an ultra high pressure unit is depicted but not required). The zoom-in is a cut-away symbolizing different peptide bands being separated in the column by different colors. Downstream, the column is connected to the rotor valve from A. The output lines feed into tubes that are filled in turn, according to the concatenation scheme. The spider-like appearance of the output lines give the name to the device. The arrows indicate that the output lines can be moved to a new set of tubes for a new separation process. After separation, the tubes are inserted into the autosampler of an UHPLC for LC-MS/MS analysis of the fractions. C, photo of the prototype spider fractionator used in this work.
Mentions: The principle of the fractionator is depicted in Fig. 1. The post-column flow from the first dimension separation enters the input port of an eight-port flow-selector rotor valve. At pre-determined time intervals, the valve switches to a new output port. Each of the outputs is connected via a narrow bore capillary to different output lines in a sample collection device, distributing the sample flow into consecutive tubes for the pooled fractions. Once one cycle has been completed, the valves switches back to the first output port and the next fluid volume is added to the already collected first fraction. In this way, the device automatically concatenates and pools the samples, without requiring different collection tubes or the combination of separately collected effluent volumes. Therefore, the volumes are not constrained to a minimum size, which would otherwise be necessary to handle them in separate tubes. We routinely employ a 250 μm inner diameter column in the first dimension at 2 μl/min and switch the valve every 90 s, thus concatenation volumes are only 3 μl. The system is fully programmable, allowing collection not only into multiples of the eight output channels (A–H) but also into as few as two or as many fractions as there are collection tubes in the device (96 in our setup). Furthermore, an arbitrary number of samples can be fractionated, and the rotor valve shifts can be defined by the user. For example, 12 samples could be scheduled for concatenation into eight fractions each in a total of 24 h using 80 min gradients.

View Article: PubMed Central - PubMed

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus