1. Krutchinsky AN, Cohen H, Chait BT
Novel Linear Ion Trap for Mass Spectrometry, US patent application 20070045533, March 1, 2007
2. Chernushevich IV, Loboda A, Thomson B, Krutchinsky AN
Mass Spectrometer Multiple Device Interface for Configuration of Multiple Devices, US Patent Application 20070057178, March 15, 2007.
3. Krutchinsky AN, Chait BT
High capacity and scanning speed system for sample handling and analysis US 7,012,249, March 14, 2006
4. Krutchinsky AN, Chait BT, Kalkum M, Cohen H, Shermann V
Method of transmitting ions for mass spectroscopy US 6,809,318, October 26, 2004
5. Krutchinsky AN, Loboda AV, Spicer VL, Ens W, Standing KG
Spectrometer provided with pulsed ion source and transmission device to damp ion motion and method of use. US 6,680,475 January 20, 2004.
6. Krutchinsky AN, Chait BT, Kalkum M, Cohen H, Shermann V
Method and system for mass spectrometry, US 6,617,577; September 9, 2003
7. Krtutchinsky AN and Chait BT
A method for increasing the signal to noise ratio of MALDI mass spectra, US 6,610,976, August 26, 2003
8. Krutchinsky AN, Loboda AV, Spicer VL, Ens W, Standing KG
Spectrometer Provided with pulsed Ion Source and Transmission Device to Damp Ion Motion and Method of Use. US 6,331,702, December 18, 2001
A protocol of coupling Ab to the magnetic Epoxy Dynal beads M270
This protocol is adapted from the original protool from Invitrogen and from the protocol used in Brian Chait’s lab at the Rockefeller University. The protocol is optimized for coupling 50 mg of epoxy Dynal beads to 500 ug of Ab. This amount of beads is usually enough for 5-10 IP experiments.
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A protocol for purification of proteins tagged with a 3xFLAG-6xH double affinity tag
1. Grow X liters of cells overnight to the density 2-5×10e7 cells/ml
2. Spin down cells in 1 liter bottles at 5000rpm 15-20’ 4ºC
3. Remove supernatant and collect cells with up to 50ml of ice cold water into 50ml conical tube.
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A protocol of IP from mammalian cell cultures
1.Culture 20 flasks (Corning, 430641; 75 cm2 culture area/flask) of mammalian cells until reach over 95% confluent.
2.Remove the culture medium, and rinse the cells twice with cold 1X PBS and aspirate.
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Cryogenic disruption of yeast cells
This is a protocol for cryogenic disruption of cells. Our protocol is a modification of the protocol designed by Markus Kalkum. Also, here you can find the the original paper first describing the use of cryogenic disruption of bacteria cells.
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A protocol for using Modular MS tool
This protocol described the major steps in using two mass spectrometers prOTOF and vMALDI-LTQ for measurement of MS, MS/MS and MSn spectra. The analysis starts with obtaining highly accurate single-stage MALDI spectra in the prOTOF mass spectrometer. Then, the MS/MS and MSn spectra of all detected species can be obtained in the vMALDI-LTQ mass spectrometer.
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In gel digestion protocol
This protocol describes a procedure for identification of proteins separated by SDS-PAGE. This protocol is based on several “classical” protocols and optimized for the efficient extraction and characterization of the tryptic peptides.
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On beads digestion protocol
This protocol describes a procedure for identification of proteins purified using 3xFLAG-6xH or 3xMyc-8xH double affinity tags. Protein and protein complexes can be directly digested on the metal chelating resin. The proteolytic peptides can be analyzed in our modular MS tool or in an HPLC-MS&MS/MS tandem mass spectrometer.
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I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions
This protocol describes the I-DIRT technique that helps to distinguish contaminants from the real interactors in immunopurified complexes. The technique was designed by Alan Tackett when he worked at Brian Chait’s Lab at the Rockefeller University.
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Vectors for tagging the protein with a florescent protein fused to a double affinity tag
We developed several vectors for tagging the proteins with a modular tag. The tag contains one of the versions of a monomeric fluorescent protein (GFP (S65T), EYFP, mOrange, mCitrine, Cherry etc.) fused at the C-terminus to a small double affinity tag for immunopurification (3xFLAG-6xH, 3xMyc-8xH, 4Strep-8xH, etc). Here are the sequences of vectors we have:
We use several mass spectrometers in the lab, a MALDI orthogonal time-of-flight mass spectrometer prOTOF (PerkinElmer), a MALDI ion trap mass spectrometer vMALDI-LTQ (Thermo Finnigan), and an electrospray QTRAP mass spectrometer (Sciex) coupled to a nano-HPLC system.
Modular Mass Spectrometric Tool
We combined two high-performance MALDI mass spectrometers, prOTOF and vMALDI-LTQ, into one mass spectrometric tool capable of providing fast, accurate, and sensitive analysis of complex biological samples.
The analysis of a typical sample starts with acquisition of a single-stage MS spectrum using the prOTOF instrument . This is a high sensitivity and high resolution device, which provides accurate, within 5-10 parts-per-million values of the masses of the detected species. Mass values extracted from the MS spectrum are used to generate an instrument control script for subsequent acquisition of fragmentation spectra from all species detect in the single-stage spectrum. The MALDI target is then transfered into the vMALDI-LTQ, an instrument optimized for very high sensitivity fragmentation analysis. This instrument is capable of fast , ~3 sec per spectrum, acquisition of MS/MS spectra in the single femtomole regime, allowing generation of 1200 spectra per hour. The successful analysis of complex peptide mixtures frequently depends on measurements of both high accuracy precursor ion masses and comprehensive fragmentation data. We achieve these properties through the combined use of two physically decoupled instruments, each optimized for a specific role. In combination, these spectrometers provide data containing 5-10 ppm accuracy in precursor mass measurement and informative fragmentation spectra while readily functioning in the single femtomole range and requiring only 2-3 seconds per MS/MS spectrum. An example illustrating the application of the modular MS tool for the analysis of the composition and phospnorylation of yeast anaphase promoting complex (APC) can be found in our publications.