Weill Medical College has established a mass spectrometry (MS) core facility for analysis of protein structure. Routinely, MS can be used for precise determination of peptide/protein molecular mass as well as to identify contaminating impurities. Methods can be employed to identify unknown proteins and often to elucidate a protein's multimeric status, posttranslational modifications, and sites of non-covalent association with small molecules or other proteins. Our MS core seeks to adapt evolving MS technologies to assist in tackling unique research problems that require protein structure analysis and identification.

The MS core, under the direction of Dr. Steven Gross, was made possible by a series of shared instrument grant awards from the National Center for Research Resources of the NIH. The core supervisor, Dr. Ivan Haller, has a solid physical chemistry background and extensive mass spectrometric experience. He assists WMC users by advising on sample preparation, performing MS analyses, and interpreting MS data. An oversight committee, comprised of WMC faculty and users, periodically reviews the core to insure that it provides optimal benefit to users.

Dr. Steven Gross

Our Location

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






Facilities

This MS Core lab in D-412 has two types of mass spectrometers that provide complementary information on protein structure. These mass spectrometers are capable of Matrix-Assisted Laser Desorption Ionization - Time Of Flight (MALDI-TOF) mass spectrometery and Electropray Ionization-tandem Mass Spectrometery (ESI-MS/MS). MALDI-TOF MS provides a sensitive, accurate and rapid procedure for determination of peptide/protein mass. Analyses are performed using an Applied Biosystems Voyager-DE PRO MALDI-TOF that was purchased in 2002. The instrument operates in both linear and reflectron mode and has a collision induced disassociation (CID) cell for enhanced fragmentation in post-source decay analyses. This instrument can provide mass information on peptides/proteins in the range of 500 to 200,000 Da, typically requires a few microliters of aqueous sample of < 1 µM concentration (i.e., 1 pmole), and achieves 99.8% mass accuracy. When the sample is an unknown protein, it is frequently possible to identify it by peptide fingerprinting i.e., computer-based matching of the observed peptides, following digestion with trypsin, vs. peptides predicted by in silico trypsinolysis of all known proteins from the species of origin. For samples in which the peptide/protein is known a priori, it is sometimes possible to identify sites with specific post-translational amino acid modifications. MALDI-TOF MS requires mixing samples with a matrix solution and applying it to a stainless steel sample target plate. Target plates are dried, introduced into the instrument and irradiated under high vacuum and fired at with a 337 nm nitrogen UV laser. With each laser shot, energy is absorbed by the matrix material and transferred to the sample, resulting in a plume of peptide/protein ions. The 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 time 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 ion. Mass of the unknown peptide/protein is deduced by averaging a requisite number of individual TOF measurements (20-1000, obtained by repeated laser firings at up to 20 times per second) and referenced to 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 concentrations of peptides/proteins in a mixture. Strengths of MALDI-TOF are its high sensitivity, broad mass range, relative tolerance to salts/buffers and suitability for the analysis of relatively complex mixtures.

ESI-MS/MS analyses are performed using either a triple quadrupole or ion trap type instrument. The triple quadrupole is a QUATRO-II (Waters Micromass) that was purchased in 1997. This instrument can measure mass/charge (m/z) over the range of 2 to 8,000. Since mass spectrometers measure mass to charge ratio, not molecular mass per se, a pure protein gives multiple peaks on a quadrupole MS instrument, corresponding to 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. A factor contributing to the precision of mass detection by ESI-MS/MS is that the mass of each of a proteins charge states contributes to the calculation of total mass. While the upper mass limit for a protein will depend on the number of charges it accepts, the practical limit is typically ~200,000 Da, with 99.98% mass accuracy. The ion trap MS is an Agilent XCTplus that was purchased in 2004. This instrument performs MSⁿ and is committed to high-throughput peptide sequencing. It is configured with a nanoLC system and nanospray-ESI source for sequence elucidation of low-abundance peptides (often < 100 fmol) in complex biologically-derived mixtures. 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 generated. 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 and therefore undetected in a MALDI-TOF MS experiment. Limitations of ESI-MS/MS are the need for relatively high purity protein samples and a poor tolerance for electrolytes and detergents.


Services Provided

Consultation. Each research problem is unique. In order to clearly define an approach and MS technique may be most suitable to answer a given question, an initial free consultation is provided. If MS is appropriate, issues of experiment design, sample preparation, timing and the cost for project completion will be discussed.

MALDI-TOF. Three levels of MALDI-TOF MS service are offered:

• 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 a solid or solution (µM) without significant buffer or detergent contamination. This service is intended to assess the purity of the peptide and possibly to specify contaminating species. Samples are analyzed on a single matrix and a spectrum of peptide constituents are provided to the user.
• 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 on 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 1-10 pmol/µl. For tryptic digests of a protein from an organism with a sequenced genome, an optional computer search can be performed to identify an unknown protein by peptide fingerprinting.
• 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 limiting amounts.

ESI-MS/MS. This service 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 recombinant 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 often be 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 protein-protein 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 approaches 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 information desired.


Contact

Steven Gross, PhD
Telephone: (212) 746-6257
E-mail:bioms@mail.med.cornell.edu