Barbara S. Larsen

Instrumental effects in the analysis of polymers of wide polydispersity by MALDI mass spectrometry

Abstract Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (tof) mass spectrometry (MS) has been reported to give low number average and weight average molecular weight values for polymers of wide polydispersity. Spectra of wide polydisperse polymers more closely resemble the distribution observed by size exclusion chromatography when obtained in the reflectron mode. However, the highest mass molecules in the distribution are not observed. These high-mass ions can be observed when the more abundant lower mass ions are deflected from reaching the detector. In addition, a mixture of three narrow distribution polymer standards selected to provide a wide mass distribution do not show discrimination against the high-mass molecules. These results are interpreted to suggest that the difficulty in observing accurate molecular weight distributions by MALDI-TOF-MS is at least in part attributable to instrumental limitations rather than ionization limitations.

An electrospray ion source for magnetic sector mass spectrometers.

Electro spray mass spectra of carbonic anhydrase (MW ∼ 29000) and ovalbumin (MW ∼ 45000) were obtained on a double focusing magnetic secter mass spectrometer by using a single stage of mechanical pumping in the interface between atmospheric pressure and high vacuum. Full scan spectra of lysozyme were recorded on 15 fmoles consumed. In addition, accurate mass measurement was demonstrated for peptides and proteins, and resolution in excess of 10,000 (m /△m, 10% valley) was observed. These results clearly show that high performance magnetic sector mass spectrometers can be advantageously interfaced to an atmospheric pressure electrospray ion source.

Laserspray Ionization on a Commercial Atmospheric Pressure-MALDI Mass Spectrometer Ion Source: Selecting Singly or Multiply Charged Ions

Multiply charged ions, similar to those obtained with electrospray ionization, are produced at atmospheric pressure (AP) using standard MALDI conditions of laser fluence and reflective geometry. Further, the charge state can be switched to singly charged ions nearly instantaneously by changing the voltage applied to the MALDI target plate. Under normal AP-MALDI operating conditions in which a voltage is applied to the target plate, primarily singly charged ions are observed, but at or near zero volts, highly charged ions are observed for peptides and proteins. Thus, switching between singly and multiply charged ions requires only manipulation of a single voltage. As in ESI, multiple charging, produced using the AP-MALDI source, allows compounds with molecular weights beyond the mass-to-charge limit of the mass spectrometer to be observed and improves the fragmentation relative to singly charged ions.

Fundamentals of the application of matrix-assisted laser desorption-ionization mass spectrometry to low mass poly(methylmethacrylate) polymers

Matrix-assisted laser desorption-ionization (MALDI) time of flight is shown to give a molar peak area response for isolated methylmethacrylate oligomers that have 25 and 50 repeat units when run on three different instruments in reflectron or linear mode and using three different matrix materials. In addition, fragmentation was not observed in any of the three different matrices or at higher laser power. No spectral differences were observed for syndiotactic and isotactic methylmethacrylate oligomers. These results suggest that the low most probable peak values observed for narrow distribution poly(methylmethacrylate) standards by MALDI mass spectrometry are not the result of mass discrimination or fragmentation.

The fundamentals of applying electrospray ionization mass spectrometry to low mass poly(methyl methacrylate) polymers

Electrospray ionization (ESI) is capable of ionizing many soluble polymers. The ESI spectra are complex because of overlap of the multiply charged ions of the oligomer distribution, causing current computer transform programs to fail. However, it is possible to determine the origin of the multiply charged ions, making it feasible to write a program designed to transform ESI polymer spectra. To assess the value of such a program for polymer analysis, isolated monodisperse methyl methacrylate (MMA) oligomers (25 and 50 repeat units) were used to determine molar signal response and propensity for fragmentation.The sum of the peak areas for the multiply charged MMA 50-mer was found to be only about 66% of the summed peak areas for the 25-mer for the same molar concentration. However, conversion of the multiply charged peak areas to the singly charged representations, with peak area compression taken into account, gave equal signal responses for the 25-and 50-mers. Signal response variations due to the tacticity of the MMA oligomers were not observed. Fragmentation of the MMA oligomers also was shown not to occur under normal ESI conditions. Therefore, transformation of the polymer spectra to the singly charged molecular ion distribution should allow accurate calculation of average molecular weights, polydispersity, end group mass, and repeat unit mass.

A mass spectrometric study of the structure of Sterol Carrier Protein SCP2 from ratliver

Abstract The amino acid sequence of Sterol Carrier Protein 2 (SCP 2 ) isolated from rat has been investigated. Using a novel mass spectrometric mapping approach, the C-terminus was found to be extended beyond the previously published sequence. Carbohydrate analysis of SCP 2 samples suggest the presence of tightly bound mannose oligosaccharide of 5–10 residues, although probably not in a glycoprotein linkage.

Method for the Determination of Perfluorooctanoic Acid in Air Samples Using Liquid Chromatography with Mass Spectrometry

Abstract Perfluorooctanoic acid is a completely fluorinated carbo-xylic acid that is usually used in the ammonium salt form as a processing aid in the production of many fluoropolymers and fluoroelastomers. Ammonium perfluorooctanoate readily dissociates in water to give the ammonium and perfluorooctanoate ions. Perfluorooctanoate has been reported to be present in low levels in human serum in the United States and Europe. This study reports on the development and validation of a method for the determination of perfluorooctanoic acid in air samples. This method uses the Occupational Safety and Health Administration (OSHA) Versatile Sampler (OVS) with a nominal 0.3 μ m filter and polystyrene resin sorbent (XAD-2 or XAD-4) followed by determination of the perfluorooctanoate anion by liquid chromatography mass spectrometry. The method was validated in the range of 0.474 to 47.4 μ g/m3 for a 480-L sample. Breakthrough studies showed samples could be collected at 1 L/min for 24 hours or at 15 L/min up to 8 hours without breakthrough. Extract storage stability tests showed that sample extracts in methanol remain stable in glass autosampler vials for up to 13 days following initial injection. Perfluorooctanoic acid stability on OVS tubes was unaffected at both refrigerated and ambient temperatures. The overall average retention efficiency was 92.1% with a pooled RSD95 of 5.8% at five concentration levels (0.474 μ g/m3 to 47.4 μ g/m3).

Sample preparation for high throughput accurate mass analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An automated sample preparation for high throughput accurate mass determinations by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed. Sample preparation was performed with an automated workstation and automated mass analyses were performed with a commercial MALDI-TOF mass spectrometer. The method was tested with a 41-sample library. MALDI-TOFMS was found to give the needed sensitivity, accurate mass measurement, and soft ionization necessary for structure confirmation, even of mixtures. A mass accuracy of 5 ppm or less was obtained in over 80% of known compound measurements. A mass accuracy better than 10 ppm was obtained for all measurements of known compounds. Analyses of parallel synthesis products resulted in 77% of the measurements with a mass accuracy of 5 ppm or better.

Efficient “total” extraction of perfluorooctanoate from polytetrafluoroethylene fluoropolymer

To determine the optimum conditions for the complete extraction of perfluorooctanoate (PFO) from polytetrafluoroethylene fluoropolymers, sample preparation and pressurized solvent extraction (PSE) conditions were investigated. Solvent extraction temperature, solvent residence time, relaxation time between extractions, and the effects of heating before PSE showed that methanol at 150 degrees C extraction temperature and a 12 min solvent residence time were the most efficient conditions. Preheating the polymer before extraction at 150 degrees C for 24 h significantly enhanced the quantity of PFO removed. Heating above 150 degrees C resulted in loss of PFO. PFO was determined by liquid chromatography with tandem mass spectrometry.

Identification of single stranded regions of DNA by enzymatic digestion with matrix-assisted laser desorption/ionization analysis

Elucidating structure function relationships of DNA in cellular processes requires fast, reliable methods that can be applied to picomole amounts of sample. Higher order structure can be inferred by distinguishing paired and unpaired regions. It is shown here that enzymatic digestion coupled with product analysis by matrix-assisted laser desorption ionization (MALDI) is able to identify unpaired bases within structured DNA regions. The method is demonstrated with DNA duplexes having a five nucleotide mismatch as a 5′ overhang, a 3′ overhang, and an internal loop. Exo- and endonuclease digestions are performed under solution conditions (temperature, annealing, and enzyme buffers) which promote base pairing and specific enzyme activity. For each type of mismatch, the length and sequence of the single stranded region can be inferred from MALDI spectra taken as a function of digestion time.