Newsletter Archive: Winter/Spring 1999

PART I: DESCRIPTION OF SERVICES

The MS core, under the direction of Dr. Steven Gross, was made possible by the award of a shared instrument grant from the National Center for Research Resources of the NIH (RR 11360). The core supervisor, Dr. Ivan Haller, has a physical chemistry background and extensive mass spectrometric experience. He will advise WMC users on sample preparation, perform MS analyses and interpret resulting data. An oversight committee comprised of WMC faculty and users will periodically review the quality and accessability of this service.

Location: WMC Room D-412
Telephone: 212-746-6334
Email: bioms@mail.med.cornell.edu

Steven Gross, Ph.D.

Contact Steven Gross @ 746-6257 for additional information 

Staff:
Steven S. Gross, Ph.D., Director
Ivan Haller, Ph.D., Supervisor
Albert Marcus Morrishow, Technician

Facility Description
This MS Core lab in D-412 comprises two types of mass spectrometers that provide complementary information on protein structure. These instruments are a Matrix Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectrometer and an ElectroSpray Ionization-tandem Mass Spectrometer (ESI-MS/MS). 

MALDI-TOF MS service is currently available and offers a sensitive, accurate and rapid procedure for determination of peptide/protein mass. Analyses are performed using a VG TofSpec (manufactured by Micromass, UK, a subsidiary of Waters, US), with a reflectron option, DEC VAXstation computer and OPUS software package. This instrument can provide mass inforrmation on peptides/proteins in the range of 500 to 200,000 Da, typically requires a few microliters of aqueous sample of <= 5 µM concentration (i.e., approximately 10 pmoles) and achieves 99.8% mass accuracy. When the sample is an enzymatic digest, it is frequently possible to identify an unknown protein constituent by computerized comparison of the clipped segments against a database of predicted fragments from all known proteins. For samples in which the peptide/protein is known a priori, it is sometimes possible to determine multimeric status and identify specific post-translational chemical modifications on proteolytic fragments. 

MALDI-TOF MS involves mixing samples with a matrix solution and applying it to a stainless steel sample disk. Sample disks are dried, introduced into the instrument and irradiated under high vacuum using a nitrogen UV laser at 337 nm. With each laser shot, energy is absorbed by the matrix material and a plume of matrix molecules carrying with it peptide/protein ions is generated. Ions are then accelerated by a strong electrical field (20-25K Volts) and travel through a field-free region toward a detector plate. The interval from firing the laser until the resulting ions strike the detector (TOF) is a function of mass to charge ratio for each given peptide/protein-derived species. Thus, accurate mass of an unknown is obtained by averaging a requisite number of individual TOF measurements (20-50, obtained by repeated laser firings at ~1 s intervals) and reference with calibration standards of known mass. A limitation of MALDI/TOF is that the measured signal intensity is not linear with the quantity of introduced sample, and therefore the method is not applicable for determining relative concentrations of peptides/proteins in a mixture. Strengths of MALDI-TOF are its high sensitivity, broad mass range, tolerance to millimolar salt concentrations and suitability for the analysis of relatively complex mixtures. 

The ESI-MS/MS, service utilizes a QUATRO-II (Micromass, UK) which has been manufactured/modified to detect an extended mass range, from 2 to 8,000 Da (contrasted with the usual 4,000 Da upper limit). Since the instrument measures mass to charge ratio rather than molecular mass per se, the protein yields a series of peaks, one for each of its charge states. Thus, a 100kDa protein with 20 of its basic sites protonated will appear as a peak with an apparent mass of 5,000 Da. One factor contributing to the precision of mass detection by ESI-MS/MS is that the mass of peaks representing each of a proteins charge states are input to the mass spectrom interpretation. While the upper mass limit for a protein will depend on the number of charges it accepts, the practical limit of the WMC MS Core Facility ESI-MS/MS should be approximately 200,000 Da with 99.98% mass accuracy. 

Electrospray ionization (ESI) introduces desolvated ions into the high vacuum environment required for mass spectrometry, from an atmospheric-pressure stream of droplets of polar molecules in a mixed aqueous/organic solvent. The stream of droplets is generated by passing the output of a syringe pump or the eluent of an HPLC through a fine stainless steel tip held at a high voltage. The triple quadrupole mass spectrometer (MS/MS) consists of two quadrupole mass analyzers separated by a collision cell. This configuration allows mass spectral analysis either directly on the ions originating in the ESI ion source (MS mode), or of the structural information-containing product ions generated by controlled fragmentation in the collision cell (MS/MS mode). ESI-MS/MS offers definitive information about protein structure; in some cases amino acid sequence and specific chemical modifications can be unequivocally deduced from the pattern of product ions generatedd. Additionally, since the technique uses soft ionization, it is possible to observe labile species, e.g., nitrosothiols, protein multimers and even biologically native non-covalent interactions, which would be destroyed in a MALDI-TOF MS and hence, undetectable. Limitations of ESI-MS/MS are the need for relatively high purity samples and a poor tolerance for electrolytes and detergents. 

ORGANIZATION AND SERVICES PROVIDED:

1. Consultation. Each research problem is unique. In order to clearly define your research problem and to determine which MS technique may be most suitable to address this problem, an initial free consultation will be provided. If MS is appropriate, issues of experiment design, instrument limitations, sample preparation and timing and cost for project completion will be discussed. 
2. MALDI-TOF. Three levels of MALDI-TOF MS service are offered:
1. Routine analysis of a synthetic peptide. This service is for analysis of peptide molecular mass (crude synthetic or purified), where the user provides 10 pmol of peptide as solid or in micromolar solution without significant buffer or detergent. It is intended to assess the purity of the known peptide and to identify specific contaminating peptide species. Samples are analyzed on a single matrix and a non-quantitative analysis of peptide constituents are provided to the user. For a peptide of 3,000 Da, mass precision by MALDI-TOF MS in reflectron mode is predictably < Da. 
2. Routine molecular weight determination of protein/peptide unknowns. This service is intended to determine the accurate molecular mass of a purified/semi-purified unknown protein or derived proteolytic fragments. Samples are analyzed using up to three different matrices to enhance the detection of protein/peptide constituents. Samples should be in 3-5 µl of water or low salt-containing buffer, preferably at a concentration of approximately 10 pmol/µl. For tryptic digests, an optional computer search can be performed where it is desirable to identify an unknown protein vs. the database of known proteins. 
3. Special handling analysis of protein/peptide unknowns. This service is intended for research problems that require special attention and effort. It will be necessary in cases where resolution and/or detection sensitivity must be optimized, such as for the detection of low molecular mass protein modifications or when samples suffer from impurities or are available in restrictive quantities.
3. ESI-MS/MS. This service offers enhanced precision for more accurate molecular mass determination of peptides/proteins (precision up to 5x that with MALDI-TOF MS), and perhaps most importantly, affords an opportunity for protein structure determinations based on the pattern of product ions produced upon fragmentation of the species of interest. Routinely, ESI-MS/MS is used to verify the predicted sequence of an expressed protein (and to identify sites of unintended mutations), as well as to quantify the extent of chemical modifications introduced into a purified protein in vitro (e.g., phosphorylation, biotinylation, fluorescein labeling). Structural information on low mass non-volatile polar compounds (synthetic or natural) can also be routinely provided. Non-routine application of ESI-MS/MS include identification of the nature and sites of in vivo protein modifications, protein multimeric status, cysteine residues engaged in disulfide bonds, amino acid sequence analysis and non-covalent interactions. It is also useful in microheterogeneity studies of isolated proteins, and can provide some tertiary structure information on native and partially denatured proteins. It should be appreciated that these non-routine applications of ESI-MS/MS utilize cutting-edge technologies that may be successfully applied in some, but not all cases. ESI-MS/MS samples should be provided in volatile polar/aqueous solvents containing less than 500 µM salt. Necessary protein quantities may range from 20-500 pmol, depending on the specific structural information desired. 

PART II: FEES FOR SERVICE

Please note that insider fees are subsidized and offered at a special introductory rate.

 

INSTRUMENT	SERVICE	WMC	Outside academic	Outside non-academic
MALDI-TOF	Synthetic peptide analysis of mass accuracy (no internal calibrant)	$25	$40	$50
	Unknown peptide/protein mass determination (with and without internal calibrant)	$35	$50	$75
	Special handling of peptide/protein unknowns (e.g., limiting sample quantity, special resolution requirements)	$50	$75	$100
ESI-MS/MS	Unknown peptide/protein mass determination	$75	$110	$150
	HPLC-MS/MS	$125	$175	$200
	Protein structure analysis/hr (multimeric status, chemical and biological modifications, amino acid sequencing, non-covalent interactions)	$80	$150	$200

PART III: Procedures for Use of the MS Core Facility
Sample submissions should be preceded by an inquiry to discuss users needs and to confirm that quantity and composition of samples are adequate for analysis. If necessary, a free consultation will be scheduled at this time. Samples are submitted with an appended "MS Core Sample Analysis Request Form", (click here to download the form in Word) providing the necessary contact, sample and billing information. 

Contact:
Dr. Steven Gross, 746-6257
bioms@mail.med.cornell.edu

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