Major Instruments

Dr. Michael DeMott and Dr. Stephen Slocum
MIT Building 16 Room 720

LC/MS: Triple Quadrupole Mass Spectrometer

These instruments are ideal for targeted low-level quantification of compounds that ionize well in ESI (electrospray ionization) mode. ESI ionization efficiency is compound dependent, but for analytes that ionize well, the sensitivity can be high; routine detection levels of biomarkers of DNA or RNA modifications, for example, are in the low femtomoles. The basic technique is 'multiple-reaction monitoring' (MRM). For MRM experiments, ions of interest are selected in the first quadrupole (Q1), fragmented in the second quadrupole (Q2), and then fragment ions of choice are selected in the third (Q3). The two filters for chemical noise (in Q1 and Q3) usually result in a significant gain in signal-to-noise and consequent lower detection limits.

The Core facility in 16-720 houses two triple quadrupole mass spectrometers, an Agilent 6410 and an API3000, both with positive and negative ion capabilities.

Online resources:

LC/MS: Quadrupole Time-of Flight (QTOF)

Quadrupole time-of-flights are high resolution instruments with mass accuracies in the low ppm range and can provide accurate masses for both molecular ions and fragment ions. For lower-molecular-weight analytes (e.g., below 600 da or so) it is usually possible to obtain reliable empirical formulas. For smaller proteins or nucleic-acid oligomers, the molecular weights can often be determined to within a dalton. These instruments are used extensively for proteomics, metabolomics, and biomarker discovery.
The Core has two QTOF systems: Agilent 6510 and Sciex QSTAR XL.
The Agilent 6510 system has three different LC configurations:

  1. Agilent 1200 capillary LC system for 'standard' conditions, i.e., flow-rates ranging from 20 to 500/minute and columns with diameters from around .3 mm to 4 mm.
  2. Agilent 1200 capillary LC and Agilent 1260 Chip Cube interface. The latter configuration uses automatically-loaded microfluidic chips with on-chip trapping and analytical columns at flow rates in nL/minute ranges. It is primarily used for MS/MS analyses of protein digests.
  3. UPLC with an Agilent 1290 Infinity for fast chromatography.

The QSTAR-XL can be operated with normal flows as well as nano flows (Agilent 1100 capillary system and 1200 nano pump).

Additional information:

LC/MS: Ion Trap

An Agilent 1200 Capillary LC interfaced with an Agilent 6340 Ion Trap. Ion traps are very sensitive in scan mode and can be used instead of single quadrupole instruments for determination of nominal molecular weights, with the additional benefit of product-ion spectra. This system, in addition, is capable of multi-stage MS/MS experiments (MSn), which can be especially useful for structure elucidation of unknowns. In MSn analyses, an ion of interest is isolated and fragmented, and the product-ion spectra recorded. One of the fragments can then be selected and fragmented in turn, and this process can be repeated as long as there are enough ions to work with. The overall result is, essentially, a sequential dissection of the original structure. As just implied, though, this technique requires a relatively large amount of sample.
Ion traps are often used to generate peptide product-ion spectra in proteomics experiments, and are also useful for analyzing nucleic acid oligomers, nucleosides, nucleotides, and small molecules; they are important in this latter context for generating targeted product-ion spectra in metabolomics experiments.
The 6340 is especially useful for analysis of post-translational modifications of proteins by using electron-transfer dissociation (ETD), which is a softer ionization mode that minimizes loss of the modifications during fragmentation and helps locate the sites of modification.

LC/MSD: Single Quadrupole

This is a user-friendly combination of an Agilent 1100 LC and an Agilent single quadrupole mass spectrometer. As with the triple quadrupoles and the ion-trap, it has lower resolution and provides only nominal molecular weights. It is, however, robust and easy to use, so most preliminary studies, e.g., routine molecular-weight determinations, method development, and reaction progress evaluations are done with this system.

LC/MS: Electrospray time-of-flight

This ESI time-of-flight systen has ion sources for standard and micro-electroscpray and atmospheric-pressure chemical ionization. Sample introduction is via syringe pump or dedicated Agilent 1200 UHPLC with diode-array detector and themostatted autosampler. This instrument has resolution typically >15K, with 1ppm mass accuracy, to determine empirical formulas for small molecules and accurate molecular weights for intact proteins and medium-sized nucleic acid oligomers. It is used as part of our standard metabolomics workflow for initial exact-mass screening. In conjunction with Mass Profile Professional software to highligh changes, along with online services such as METLIN, it provides targets for MS/MS experiments to confirm structures and to access pathway analysis software such at the Mass Profiler Professional add-on or KEGG. Located in 56-747.


For Online Resources:

GC/MS: Agilent 5973N

EI (electron ionization), PCI (positive chemical ionization) and NICI (negative ion chemical Ionization).
In general, GC/MS is useful for analyzing volatile organic compounds, e.g., aliphatic hydrocarbons, polyaromatic hydrocarbons up to MW 300, pesticides , herbicides, fatty acids (after conversion to esters), and amino acids after derivatizing the acid and amine groups.
EI: After separation in the GC, molecules are ionized and fragmented with 70EV electrons. This energy is standard across virtually all GC/MS platforms, and EI Mass spectra are highly reproducible from laboratory to laboratory and therefore searchable against NIST/EPA/NIH mass spectral libraries, which significantly helps with identification of unknowns.
The relatively high energy of EI often produces mass spectra where the molecular ions are absent or of very low abundance. PCI and NICI sources operate with higher pressures of a reagent gas (usually methane) where the electron beam ionizes the reagent gas which then transfers charge to the analytes. These are much softer ionization methods that can produce molecular ions with much less fragmentation, and are therefore strongly complementary with EI.
PCI is thus used primarily for fragile molecules that do not yield molecular ions with EI. In addition, since the charge is distributed over fewer fragment ions, this often helps with sensitivity and detection of trace organic compounds.
NICI is the method of choice for detection and quantification of molecules with strongly electronegative substituents, e.g. F, Cl, I, Br or NO2 groups. Compounds are often derivatized with highly fluorinated derivatizing agents to enhance sensitivity in NICI.

ICP-MS: Agilent 7900


Agilent 7900 ICP-MS has a robust plasma with the new orthogonal detector system (ODS) which provide a wide dynamic range (up to 11 order of magnitude dynamic range) from sub-ppt to percent-level concentration therefore trace elements can be measure along with high concentration elements in the same run.
With the combination of the Milestone UltraWave microwave sample-digestion system, the CEHS core facility has capacity of analyzing geological samples. The CEHS core facility now has trace metal speciation capabilities by having a dedicated HPLC system for the Agilent ICP-MS system.
Prior training with Dr. Stephen Slocum is necessary before using the Agilent 7900 ICP-MS or the Milestone UltraWave microwave. Please contact Dr. Stephen Slocum (via the online inquiry form) to schedule a training time.

Liquid Chromatography

In addition to the LC/MS instruments listed above, the Core has two free-standing Agilent 1100 HPLC systems, both with diode array detectors (DAD). One is dedicated to method development, identification and quantification. The other, with a fraction collector, is primarily used for fractionation of trace biomarkers of DNA and RNA modifications, but could be configured for separations of other mixtures.