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What is Mass Spectrometry?

For our purposes, mass spectrometry is an analytical technique by which we measure the m/z or mass/charge ratio of charged particles we've introduced by use of our electrospray ionization ESI from the liquid phase, allowing for liquid chromatography to occur in-line with the instrumentation. It is nearly that simple. However, in application, there are many factors that must be considered to achieve desired results.

Nano-electrospray ionization (NSI)

Nano-ESI is essentially the same process as conventional ESI with a primary difference in the flow rates involved. ESI is conventionally performed at μL/min flow rates and involves the coordination of gas flows around the spray tip. Nanospray is typically performed at < 1 μL flow rates, to as low as ~30 nL/min.

Ultra-High Pressure/Performance Liquid Chromatography (UPLC)

To provide ever better chromatographic separations, instrumentation has gone to ever increasing pressures. For NSI applications, the low flow rates and desire to limit the 'dead volume' or space in which chromatographic resolution might be lost by diffusion, the entire system is very low volume. Because of these concerns, tubing is conventionally fused-silica with a very small internal diameter ( < 100 microns). Chromatography is performed in columns packed inside this glass tubing, and with the smallest beads possible. These factors result in a very high pressure due to the small diameters and flow restriction by these beads.

Fragmentation

In any given sample, many different peptides can produce a mass that, even with our typical <2 ppm mass accuracy, is not sufficient to determine the identity of the peptide. Similar issues exist in lipid and metabolite samples. Fragmentation provides additional diagnostic details to assist in identification of the precursor mass, even definitively. For further information, visit this site, which focuses on identification but also explains fragmentation well. De Novo Sequencing

Fragmentation is performed on an isolated ion. Hence, a recent (and thus pertinent to the current ions) primary scan lets us determine precursor m/z values for which fragmentation might be performed. A fresh batch of ions is then collected in the ion trap and modulation of the trap results in the ejection of ions outside of the (typically) 1 m/z range around the chosen precursor mass and retention of the chosen ion with > 98% efficiency. Fragmentation can then be performed.

As fragmentation is essentially a gas phase reaction, there are a number of methods employed that vary in the fragmentation efficiency and in specificity for specific cleavage sites. To that end, using the appropriate fragmentation method for the contents of a sample and the information for which you are looking is an important decision in experimental design. Hence, here is presented a brief outline of the fragmentation methods of which this instrument is capable.

Collision Induced Dissociation (CID)

CID is the first and most typical fragmentation method. Essentially, CID employs an RF excitation of the trapped ion cloud. This ion cloud moves much more rapidly under this excitation and collides with Helium gas that is allowed into the trap area. Hence, the excitation energy can be modulated by changing the RF field applied to the ions. CID results in cleavage of the protein backbone in a characteristic series of ions labeled as b and y ions. These indicate which ion you are seeing from a cleavage of the peptide backbone just C-terminal of the carbonyl of each amino acid, the amide bond with the amine group.

CID is the most well characterized cleavage and the algorithms used for predicting the cleavages induced (crucial for identification by current 'gold standard' methods) are most accurate. CID as a reaction has downsides that are currently offset by the superior matching of the algorithms. CID produces a kinetic type reaction that results in cleavage of the most labile bonds. Hence, this produces preferential cleavages of weaker bonds such as phosphoesters formed by post-translational phosphorylation of serine, threonine or tyrosine residues.

CID is robust and capable of adequate fragmentation for most uses. It is the fastest fragmentation method allowing for the greatest number of secondary spectra per second and is usually sufficient for peptide identification.

Higher-energy C-trap Dissociation (HCD)

HCD is basically CID on steriods. It involves shooting the ion cloud into a direction ion optics tube where nitrogen floats around. Because of the higher energies involved, HCD results in more cleavage and doesn't suffer from the rearrangement issues of preferential cleavages exhibited by CID fragmentation.

Downsides... FT analysis required in Orbitrap.

Electron-Transfer Dissociation (ETD)

The bomb diggity, but has some reliability issues and such...

Data Analysis

Matlab serves as an excellent resource in learning the basic data structure of hyphenated mass spectrometry methods. Reading and participating in their demos are a MUST if you are serious about getting up to speed on terminology, challenges, and general strategy in handling MS data. Entering this field requires lots of time, but you are lucky that Matlab has amazing documentation. If you have never used Matlab before, know that it has superb documentation to walk you through; it's replete with examples and explanations that outclasses many alternative development tools.

Finding Demos

Running Demos

Our Instrumentation

We run a Thermo-Fischer LTQ Orbitrap XL w/ ETD coupled to a Eksigent Ultra 2D LC system capable of maximum pressures of 10,000 psi and flow rates on the two pumps of < 1000 nL/min and < 30 μL/min (discuss the two pumps)...

Guidelines for use

We are not lab technicians. Unless that changes, we are unable to allow for unorthodox uses of the instrument. Contact us if you have other questions.

Criteria for use

There are two types of samples we are willing to run. Please understand that determining what a spectrum represents can be very challenging. For your own sake, we require certain criteria for samples so that we can provide some consistency in understanding how to improve results. There are two particular concerns with samples. One is ion suppression that results from having contaminants that are easily ionized in a sufficient concentration to reduce the ionization of your analyte. Of particular concern here are both salts (which can also 'crash' out of solution and clog our system) and ionic detergents. For these reasons, we will not run any sample containing more than 50 mM salts or any ionic detergents. We have ways of eliminating these issues through sample prep, as here outlined, but for further questions, come and visit with Aman or Ryan.

Direct Injection

Ordering Information

Qty Unit size Description Catalog No Cost
1 ea Teflon Tubing sleeve, 395 μm ID and 1/16" OD, Green F-242x $15.00
1 ea Adapter, quick connect female luer-lok to female 10-32 coned fitting P-659 $15.00
1 ea One-piece, fingertight 1/16" 10-32 fitting F-120 $15.00
1 ea Fused silica, 20 μm x 360 μm x 2m FS-120 $45.00

If you will buy these parts, you won't have to worry about potential contamination from anything else that might have been run through our syringes. It benefits you.

LCMS

Parts/Supplies Ordering information

Scheduling

You may access the scheduling site here:

Orbitrap Scheduling