LC-MS/MS  @ Yang Lab

Boston VA Research Institute

150 S. Huntington Ave. 1A

Boston, MA 02130   
Jinghua.yang@va.gov

Tel: (857) 364-5611

    Fax: (857)364-5627                                               

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1. Nano-LC QSTAR® Elite Hybrid LC/MS/MS System

The QSTAR® Elite System coupled with the Tempo nano LC System provides rapid, robust protein identification. The QSTAR Elite LC/MS/MS system is a Q-TOF mass spectrometer for proteomics and metabolomics applications. The QSTAR Elite has improved sensitivity, acquisition speed, resolution, dynamic range, and mass-measurement accuracy for more-confident identifications. The system includes ProteinPilot software can be used for iTRAQ and SILAC analyses. The instrument is equipped with the Tempo LC system as a fully integrated LC/MS/MS solution for biomarker discovery.

 

 

 

2. Voyager-DE™ STR Biospectrometry Workstation

 The MALDI-TOF mass spectrometer is an Applied Biosystems VOYAGER-DE STR workstation. This is a matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer with both linear and reflectron modes providing excellent resolution, sensitivity, and mass accuracy. Clients can choose from positive or negative ion detection modes of operation as well as the ability to perform Post Source Decay (PSD) experiments to obtain valuable peptide sequence information.

 

 

 

 

 

 

 

Applications:

LC-MS/MS is now a standard method for the analysis of protein samples that have been separated by 1D or 2D gel electrophoresis with subsequent in-gel digestion. However, the throughput tends to be low for this workflow since the analysis typically requires the use of longer gradients and data acquisition times of over 30 minutes per sample. Here we show very rapid LC gradients – as fast as 1 minute –using monolithic columns combined with intelligent, efficient data acquisition to provide protein identification results that are equivalent to standard gradient separations.

The Tempo™ nano LC system employ a direct pumping system with independent flow rate monitoring for each mobile phase, and is capable of delivering a precision of <1 nL/min before mixing. This enables precise, rapid gradient formation. Close coupling of the pump, gradient mixer and autosampler minimizes dispersion and delay time enabling very fast LC runs.

Sample: Bovine serum albumin (Michrom BioResources) was digested with trypsin and injected at a level of 100 fmol of digest on column.

Chromatography: Very rapid nanoflow chromatography was performed using the Tempo™ nanoLC system. A Chromolith column (Merck, 100 μm ID) was run using a 1 minute gradient at a flow rate of 800 nL/min. The total LC method time including re-equilibration from injection to injection was 6 minutes (Figure 2).

Mass Spectrometry: LC/MS/MS analysis was performed using the NanoSpray® II source and Heated Interface on the QSTAR ® Elite LC/MS/MS System. Using the new Smart IDA (Information Dependent Acquisition) features within Analyst® QS 2.0 Software, extremely fast and intelligent MS/MS acquisitions were performed.

Database Searching: Protein identification was performed using the Paragon™ Algorithm in ProteinPilot™ Software or Mascot. Sequence coverage was automatically calculated within the software.