Sensitivity Of Matrix-assisted Laser Research Articles

Synthesis and end-group control of soluble, high-molecular-weight hyperbranched poly(phenylene thienylene)s by non-stoichiometric A2 + B3 Suzuki-Miyaura polycondensation without gelation

Non-stoichiometric Suzuki-Miyaura polycondensation of tribromo monomer 1, containing a thienylene group, with diboronic acid (ester) monomer 7 in the presence of tBuPPd catalyst, which has a propensity for intramolecular catalyst transfer on a π-electron face, was investigated. We anticipated that the presence of thienylene groups in the obtained hyperbranched polymer would increase the detection sensitivity in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and enable the analysis of the polymer end groups up to the high-molecular-weight region. We first conducted Suzuki-Miyaura reaction of several kinds of 1 with phenylboronic acid 2 in the presence of tBuPPd precatalyst 3. The reaction of tris(5-bromo-4-hexylthien-2-yl)benzene 1c with 2 afforded exclusively tri-substituted product 4c, indicating that the tBuPPd catalyst undergoes intramolecular catalyst transfer on 1c after the first and second substitutions of 1c with 2. The polycondensation of equimolar 1c and pinacol phenylenediboronate 7a (reaction site ratio of Br:BPin = 1.5:1.0 (BPin = pinacol boronate)) was then carried out in the presence of 3 for 3 h, affording hyperbranched polymer with Mn = 37500 without gelation. The product was purified by precipitation. The degree of polymerization (DP) was calculated from the absolute molecular weight, determined by multi-angle light scattering (MALLS) analysis, to be 200, which is a far higher than would be predicted from the Flory-Stockmayer theory at the critical gel point (DP = 6.6). Furthermore, the MALDI-TOF mass spectra showed two series of peaks due to polymer with boronate ends and boronate ends along with one cyclic unit up to m/z of about 7500, as we had expected. The absence of peaks due to polymer with Br ends indicates that the degree of branching (DB) of this hyperbranched polymer is 100 %.

3D ToF-SIMS Analysis of Peptide Incorporation into MALDI Matrix Crystals with Sub-micrometer Resolution.

The analytical sensitivity in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is largely affected by the specific analyte-matrix interaction, in particular by the possible incorporation of the analytes into crystalline MALDI matrices. Here we used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to visualize the incorporation of three peptides with different hydrophobicities, bradykinin, Substance P, and vasopressin, into two classic MALDI matrices, 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA). For depth profiling, an Ar cluster ion beam was used to gradually sputter through the matrix crystals without causing significant degradation of matrix or biomolecules. A pulsed Bi3 ion cluster beam was used to image the lateral analyte distribution in the center of the sputter crater. Using this dual beam technique, the 3D distribution of the analytes and spatial segregation effects within the matrix crystals were imaged with sub-μm resolution. The technique could in the future enable matrix-enhanced (ME)-ToF-SIMS imaging of peptides in tissue slices at ultra-high resolution. Graphical Abstract ᅟ.

The effect of laser profile, fluence, and spot size on sensitivity in orthogonal‐injection matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry

The influence of incident laser parameters on sensitivity in matrix-assisted laser desorption/ionization (MALDI) has been investigated using orthogonal-injection time-of-flight (TOF) instruments. A qualitative comparison was first made between the beam profiles obtained with a N(2) laser and a Nd:YAG laser using 2-m long optical fibers. The N(2) laser gives better sensitivity, consistent with a more uniform fluence distribution and therefore better coverage of the N(2) laser profile. Most of the difference disappears when a 30-m long fiber is used or when the fibers are twisted during irradiation to smooth out the fluence distribution. In more systematic measurements, the total integrated ion yield from a single spot (a measure of sensitivity) was found to increase rapidly with fluence to a maximum, and then saturate or decrease slightly. Thus, the optimum sensitivity is achieved at high fluence. For a fluence near threshold, the integrated yield has a steep (cubic) dependence on the spot size, but the yield saturates at higher fluence for smaller spots. The area dependence is much weaker (close to linear) for fluence values above saturation, with the result that the highest integrated yields per unit area are obtained with the smallest spot sizes. The results have particular relevance for imaging MALDI, where sensitivity and spatial resolution are important figures of merit.

A case study of de novo sequence analysis of N-sulfonated peptides by MALDI TOF/TOF mass spectrometry

The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have increased its application in recent years. The most common method of "peptide mass fingerprint" analysis often does not provide robust identification. Additional sequence information, obtained by post-source decay or collision induced dissociation, provides additional constraints for database searches. However, de novo sequencing by mass spectrometry is not yet common practice, most likely because of the difficulties associated with the interpretation of high and low energy CID spectra. Success with this type of sequencing requires full sequence coverage and demands better quality spectra than those typically used for data base searching. In this report we show that full-length de novo sequencing is possible using MALDI TOF/TOF analysis. The interpretation of MS/MS data is facilitated by N-terminal sulfonation after protection of lysine side chains (Keough et al., Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7131-7136). Reliable de novo sequence analysis has been obtained using sub-picomol quantities of peptides and peptide sequences of up to 16 amino acid residues in length have been determined. The simple, predictable fragmentation pattern allows routine de novo interpretation, either manually or using software. Characterization of the complete primary structure of a peptide is often hindered because of differences in fragmentation efficiencies and in specific fragmentation patterns for different peptides. These differences are controlled by various structural parameters including the nature of the residues present. The influence of the presence of internal Pro, acidic and basic residues on the TOF/TOF fragmentation pattern will be discussed, both for underivatized and guanidinated/sulfonated peptides.

Increasing sensitivity and decreasing spot size using an inexpensive, removable hydrophobic coating for matrix-assisted laser desorption/ionisation plates.

Spot size reduction and increased detection sensitivity in matrix-assisted laser desorption/ionisation (MALDI) of small molecules are accomplished by using an inexpensive and removable hydrophobic coating for MALDI targets, based on 3M Scotch Gard surface treatment. Several variations in sample preparation were explored, such as surface coating technique, identity of the matrix, solvent composition, and the type of metal support plate used. These were investigated on both uncoated and coated surfaces and their impact on spot size, crystal coverage, and sensitivity is presented here. Additionally, crystallisation behaviour obtained on coated plates is compared with that on uncoated plates using scanning electron microscope analysis. To demonstrate the potential of the new coating technique, erythromycin A and valinomycin are studied to determine the increase in detection sensitivity of coated plates in comparison to uncoated plates, and to reveal the suitability of the plates for application in combined high-performance liquid chromatography/MALDI (HPLC/MALDI), where widely varying solvent compositions and droplet volumes are observed. It is shown that enhancements in detection sensitivities correlate very well with the achieved spot size reduction. The versatility of the coated plates is also exhibited by the ease of removing the surface layer, after which the plates can be rigorously cleaned without worry about damaging the hydrophobic surface, followed by a quick reapplication of new hydrophobic coating material. This makes the non-polar coating superior to more expensive commercial hydrophobic-coated targets, which are much more delicate to clean. Furthermore, cleaning and reapplication eliminate potential carry-over effects and the easy application procedure also makes the fabrication of inexpensive, disposable MALDI targets readily possible.

Charge derivatization by 4-sulfophenyl isothiocyanate enhances peptide sequencing by post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

High-sensitivity, rapid identification of proteins in proteomic studies normally uses a combination of one- or two-dimensional electrophoresis together with mass spectrometry. The simplicity and sensitivity of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have increased its application in recent years. The most common method of 'peptide fingerprinting' often may not provide robust identification. Normally additional sequence information by post-source decay (PSD) MALDI-TOFMS provides additional constraints for database searches to achieve highly confident results. Here we describe a derivatization procedure to facilitate the acquisition of such sequence information. Peptide digests from a skin-expressed protein were modified with 4-sulfophenyl isothiocyanate. The resulting peptides carry a fixed negative charge at the N-terminal end and the resulting PSD spectrum is dominated by C-terminal y-type ions. The sequence information in most cases can be obtained manually or with simple programming tools. Methods of optimizing the procedure and increasing the sensitivity are discussed.

Rapid, sensitive structure analysis of oligosaccharides.

We have developed an efficient method for the derivatization of oligosaccharides, wherein the oligosaccharide is efficiently ligated to a basic aminooxyacetyl peptide by oxime formation. The resulting glycopeptide yields much higher sensitivity in matrix-assisted laser desorption/ionization mass spectrometry than does the underivatized oligosaccharide. Digestion of the glycopeptide by a exoglycosidase array and subsequent mass spectrometric assay of the digestion products provide a sensitive and rapid way to elucidate the structure of the oligosaccharide. In addition to oligosaccharide sequencing, the ligation reaction between an oligosaccharide and an aminooxyacetyl peptide also provides a potentially very convenient and efficient way for the synthesis of glycopeptides or glycoproteins.