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A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day.

Hosp F, Scheltema RA, Eberl HC, Kulak NA, Keilhauer EC, Mayr K, Mann M - Mol. Cell Proteomics (2015)

Bottom Line: The modified LC platform eliminates idle time between measurements, and the high sequencing speed of the Q Exactive HF reduces required measurement time.Thus, sample throughput, sensitivity and LC/MS-MS duty cycle are improved severalfold compared with established workflows.The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

No MeSH data available.


Related in: MedlinePlus

Parallel UHPLC operation with two analytical columns. (A) In this position of valve S, the sample pump can fill the sample loop. (B) By switching valve S, the contents of the sample loop can be loaded onto one of the analytical columns. (C) In this position of valve W, the analytical column 1 can be eluted with the mobile phase, while analytical column 2 is loaded. (D) By switching the position of valve W, this behavior is inverted. (E) In the conventional setup, the mass spectrometer is not sequencing while the HPLC is loading a new sample. The light gray arrow indicates where the mobile phase is active. (F) With the double-barrel setup, this idle-time is circumvented, enabling almost continuous operation. (G) Positioning of the analytical columns in reference to the inlet of the mass spectrometer. (H) Redesign of the column oven for two analytical columns.
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Figure 2: Parallel UHPLC operation with two analytical columns. (A) In this position of valve S, the sample pump can fill the sample loop. (B) By switching valve S, the contents of the sample loop can be loaded onto one of the analytical columns. (C) In this position of valve W, the analytical column 1 can be eluted with the mobile phase, while analytical column 2 is loaded. (D) By switching the position of valve W, this behavior is inverted. (E) In the conventional setup, the mass spectrometer is not sequencing while the HPLC is loading a new sample. The light gray arrow indicates where the mobile phase is active. (F) With the double-barrel setup, this idle-time is circumvented, enabling almost continuous operation. (G) Positioning of the analytical columns in reference to the inlet of the mass spectrometer. (H) Redesign of the column oven for two analytical columns.

Mentions: Next, we set out to develop a double-barrel chromatography system in order to reduce the idling time of the mass spectrometer during loading of the peptides to the LC column. Unfortunately, no such setup has been described for the Thermo EASY-nLC 1000 UHPLC systems (Thermo Fisher Scientific) that we employ and that are widely used with the Orbitrap-family of mass spectrometers. To address this, we modified the liquid pathway of the EASY-nLC 1000 UHPLC system (Figs. 2A-2D). In brief, we placed the sample loop directly between the pump S and valve S, allowing the system to utilize pump S as both the sample pickup as well as the sample-loading pump (in the original setup, pump A is used as sample-loading pump). The valve S is connected to valve W (in the original setup this valve is connected to a waste line used for rapid evacuation of the buffers from the lines), which connects to the buffer A and B mixing-T connection and the two analytical columns through standard sample lines. This setup allows loading of one sample onto one of the analytical columns while the other is eluted.


A Double-Barrel Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) System to Quantify 96 Interactomes per Day.

Hosp F, Scheltema RA, Eberl HC, Kulak NA, Keilhauer EC, Mayr K, Mann M - Mol. Cell Proteomics (2015)

Parallel UHPLC operation with two analytical columns. (A) In this position of valve S, the sample pump can fill the sample loop. (B) By switching valve S, the contents of the sample loop can be loaded onto one of the analytical columns. (C) In this position of valve W, the analytical column 1 can be eluted with the mobile phase, while analytical column 2 is loaded. (D) By switching the position of valve W, this behavior is inverted. (E) In the conventional setup, the mass spectrometer is not sequencing while the HPLC is loading a new sample. The light gray arrow indicates where the mobile phase is active. (F) With the double-barrel setup, this idle-time is circumvented, enabling almost continuous operation. (G) Positioning of the analytical columns in reference to the inlet of the mass spectrometer. (H) Redesign of the column oven for two analytical columns.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587330&req=5

Figure 2: Parallel UHPLC operation with two analytical columns. (A) In this position of valve S, the sample pump can fill the sample loop. (B) By switching valve S, the contents of the sample loop can be loaded onto one of the analytical columns. (C) In this position of valve W, the analytical column 1 can be eluted with the mobile phase, while analytical column 2 is loaded. (D) By switching the position of valve W, this behavior is inverted. (E) In the conventional setup, the mass spectrometer is not sequencing while the HPLC is loading a new sample. The light gray arrow indicates where the mobile phase is active. (F) With the double-barrel setup, this idle-time is circumvented, enabling almost continuous operation. (G) Positioning of the analytical columns in reference to the inlet of the mass spectrometer. (H) Redesign of the column oven for two analytical columns.
Mentions: Next, we set out to develop a double-barrel chromatography system in order to reduce the idling time of the mass spectrometer during loading of the peptides to the LC column. Unfortunately, no such setup has been described for the Thermo EASY-nLC 1000 UHPLC systems (Thermo Fisher Scientific) that we employ and that are widely used with the Orbitrap-family of mass spectrometers. To address this, we modified the liquid pathway of the EASY-nLC 1000 UHPLC system (Figs. 2A-2D). In brief, we placed the sample loop directly between the pump S and valve S, allowing the system to utilize pump S as both the sample pickup as well as the sample-loading pump (in the original setup, pump A is used as sample-loading pump). The valve S is connected to valve W (in the original setup this valve is connected to a waste line used for rapid evacuation of the buffers from the lines), which connects to the buffer A and B mixing-T connection and the two analytical columns through standard sample lines. This setup allows loading of one sample onto one of the analytical columns while the other is eluted.

Bottom Line: The modified LC platform eliminates idle time between measurements, and the high sequencing speed of the Q Exactive HF reduces required measurement time.Thus, sample throughput, sensitivity and LC/MS-MS duty cycle are improved severalfold compared with established workflows.The pipeline can be extended to different types of interaction studies and to other medium complexity proteomes.

View Article: PubMed Central - PubMed

Affiliation: From the ‡Department of Proteomics and Signal Transduction, Max-Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.

No MeSH data available.


Related in: MedlinePlus