LC/MS/MS

Protein identification and characterization using LC/MS/MS has become the workhorse of proteomics, primarily because:

Automated data acquisition and processing is routine
Complex mixtures of proteins can be analyzed
High sensitivity levels are attainable
The sites/identity of post-translational modifications can be probed

Our laboratory is equipped with a Micromass Q-Tof2 tandem mass spectrometer that we interface with nano-LC systems. Database searching is performed using Mascot.

We utilize LC/MS/MS for multiple types of analysis:

Protein identification from gels
1. When MALDI is not sensitive enough
2. When there are insufficient numbers of peptides to allow a confident ID by MALDI
3. Protein identification from SDS-PAGE bands
Protein identification from complex protein mixtures in solution.
These will often require:
1. Long gradient LC/MS/MS
2. 2D-LC/MS/MS
De novo sequencing
1. We utilize de novo sequencing when a species is poorly represented in the protein or EST databases (or the protein of interest is not yet entered), performing protein characterization studies and working with non-tryptic peptides (such as naturally occurring peptides or those generated from a different proteolytic enzyme) – here the fragmentation pathways under low energy collision-induced dissociation (CID) conditions can be difficult to rationalize and are not always accounted for by search engines.
Protein characterization – identifying sites of protein modification

LC/MS/MS Set-Up

The manufacture, use, and/or sale of V-Column technology is owned and protected by Harvard Medical School. For licensing information, please contact:

Henry F. Oettinger, PhD, MBA
Office of Technology Licensing & Industry Sponsored Research
Harvard Medical School
Direct Phone: (617) 432-1744
Fax: (617) 432-2788
e-mail: henry_oettinger@hms.harvard.edu

Recent work by the Gygi group (Peng, J. and Gygi, S.P. J. Mass Spectrom., 36, 1083 (2001)) demonstrated the use of a vented column system (V-column) to allow increased flow rates for sample loading without the use of pre-columns. Pre-columns loaded at µL/min flow rates can lead to sample loss and decreased chromatographic performance. The v-column system exploits the fact that peptides are actually bound to the first few mm of a column during direct loading; the column is “vented” after 1 cm giving a flow rate of 3 µL/min through the vent due to a reduction in the back pressure normally encountered. The vent line is connected to a six-port valve with one port open and a second closed off, switching to the closed position directs the flow back through the remainder of the column at conventional (~200 nL/min) flow rates. Both flow rates are achieved from the same pump flow rate.

Our experimental set-up is outlined in the following schematic. The initial Gygi work presented at ASMS (Licklider, L.J., Thoreen, C., Peng, J., Gerber S.A. and Gygi, S.P. Proceedings of the 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, Illinois (2001)) suggested a physical cut in the column with a frit therefore required at the entrance to the vent line. We have exploited the use of IntegraFrit columns (New Objective) for the v-column that have an internal silica frit. The IntegraFrit and subsequent PicoFrit column are packed with the same reverse phase material (Symentry, Waters). The use of a micro tee with a platinum wire facilitates voltage acquisition by the solvent.

At a 150 µL/min flow rate from the pump the flow through the PicoFrit column with the injector closed is 200 nL/min. Flow through the vent line with the injector open is 3 µL/min.

LC/MS/MS Sensitivity

The following example shows how the high sensitivity LC/MS system we use can identify proteins from the weakest visible spots isolated from 2-D gels. The gel was stained using Sypro® Ruby (Molecular Probes) and imaged using a PerkinElmer Life Sciences ProXPRESS fluorescent imager.

The expanded view of the gel image has been contrast stretched to allow visualization of the weakest gel spots. Spot number 176 was excised robotically using Investigator™ ProPic and transferred to the InvestigatorTM ProGest digest robot. Following digestion, approximately 25% of the digest supernatant (5 µL) was analyzed by high sensitivity LC/MS/MS and the data searched against NCBI using Mascot. The results are shown below.

Following trytic in-gel digestion, MS analysis, and submission of the peptide list to the NCBI database , the following match was returned:

gi|6754222 Mass: 30926 Total score: 250 Peptides matched: 6
heterogeneous nuclear ribonucleoprotein A/B [Mus musculus]

Nominal mass of protein (Mr): 30926
Fixed modifications: Carbamidomethyl (C)
Cleavage by Trypsin: cuts C-term side of KR unless next residue is P
Sequence Coverage: 21%
Calculated pI value: 7.68

Matched peptides shown in Bold Red

MSDAAEEQPM ETTGATENGH EAAPEGEAPV EPSAAAAAPA ASAGSGGGTT TAPSGNQNGA EGDQINASKN EEDAGKMFVG GLSWDTSKKD LKDYFTKFGE VVDCTIKMDP NTGRSRGFGF ILFKDSSSVE KVLDQKEHRL DGRVIDPKKA MAMKKDPVKK IFVGGLNPEA TEEKIREYFG QFGEIEAIEL PIDPKLNKRR GFVFITFKEE DPVKKVLEKK FHTVSGSKCE IKVAQPKEVY QQQQYGSGGR GNRNRGNRGS GGGQGSTNYG KSQRRGGHQN NYKPY

This is an example of the product ion data obtained:

MS/MS Fragmentation of FGEVVDCTIK
Found in gi|6754222, heterogeneous nuclear ribonucleoprotein A/B [Mus musculus]

Monoisotopic mass of neutral peptide (Mr): 1166.5767
Fixed modifications: Carbamidomethyl (C)
Ions Score: 67 Matches (Bold Red): 15/81 fragment ions using 31 most intense peaks

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