One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

An ambitious initiative is proposed to generate a definitive reference set of human proteoforms.

The block length distributions of two block copolymers of R-methylstyrene and styrene, of different molecular weights and relative block lengths, were analyzed by matrix-assisted laser desorption/ ionization time-of-flight (MALDI/TOF) mass spectrometry. A method for the treatment of mass spectroscopic data for block copolymers is proposed. The random coupling hypothesis has been verified experimentally and found to hold for this type of triblock copolymers. The experimental distributions have been compared with the Poisson and Schulz-Zimm models modified to accommodate the change of variables. The agreement between the experimental and calculated distributions is very good for the Schulz-Zimm model and fair for the Poisson model. Mass spectrometry proved to be a useful technique providing unique information on the experimental distributions of both constituent parts in a block copolymer. It was confirmed that block copolymers with narrow molecular weight distributions may have broad, complex, and even bimodal composition distributions. The polydispersity factors observed for the individual parts were higher than those for the whole copolymer. IntroductionIn the recent past, there has been a considerable upswing and interest in properties of block copolymers. Micellization, brush formation, and drug delivery are just three examples of the areas in which block copolymers have been receiving extensive attention. For the study of many of these properties, detailed information about the block lengths and compositions of each of the segments is needed. However, while it has been possible to determine the total molecular weight and the molecular weight distribution of the block copolymer, as well as the molecular weight and molecular weight distribution of the first segment to be prepared, 1,2 it has not been possible, with the exception of few cases, 3-5 to determine independently the molecular weight and molecular weight distribution of the second segment. This information can be of considerable importance, since, to pick one example, it has recently been shown that the size of the core of block copolymer micelles is very much a function of the molecular weight distribution of the core forming block. 6 In the above case, the core forming block was composed of vinylpyridinium methyl iodide, while the corona consisted of styrene. In that case, the block length distribution was broadened artificially to obtain very large values of the heterogenity index. However, in general, it has been impossible to obtain detailed data on the second block (e.g., vinylpyridine) independent of the first (e.g., styrene). A number of theoretical treatments exist which make possible the calculation of the molecular weight distribution of the second block from the molecular weight distribution of the first block and of the total block copolymers. These methods are very useful for some compositions, but, if the first block is sizable and the second block is relatively small, the error associated with the applications of this method can be app...

One of the most important factors limiting the universal application of matrix-assisted laser desorption/ionization mass spectrometry (MALDI) to analytical problems is the preparation of the analyte in a proper matrix. This problem is particularly exacerbated in working with organic-soluble polymers since nearly all matrix/solvent systems have been developed for the analysis of materials of biological interest that have significant solubility in aqueous environments.'s2 Previous work in this area has shown that water-soluble polymers can be analyzed using conditions similar to those of the biopolymer~.~ Additionally, MALDI on polygly~ols.'-~ poly(styrene),6 and poly(methylmethacrylate)6*8 have been shown using solvent mixtures and salt addition in the preparation. During the solvent removal from the matrix/analyte mixture the solubility of the polymer often is such that it will precipitate out of solution before crystallization of the matrix, preventing good conditions for MALDI. In order to ensure more interaction between the polymer and the matrix we have moved to simpler solvent systems than the typical water, acetonitrile, ethanol, etc. mixtures and will present results here showing that good polymer spectra can be obtained using the common matrices and organic solvents.All measurements were performed on the Bruker Reflex time-of-flight mass spectrometer with 30 or 35 kV acceleration and detection in either the linear or reflected mode. The samples were ionized by irradiation with the output of an N, laser (337 nm, 5 ns) with power levels slightly above threshold (-106-107 W cn-'). Low mass ions were removed with pulsed deflection and 100-200 transients were summed for the spectra shown. All matrices are from Aldrich except the 7amino-4-methylcarbostyril which is from Kodak. The poly(methy1 methacrylate) (pMMA) molecular weight stanr.i. 0.40 0.30 0.20dards are from Polymer Laboratories (28k, 59k, 127k and 260k) and American Polymer Standards Corp. (10k). The pMMA was dissolved in acetone at a concentration of 1 x lo-' M for the 260k, 2 x lo-' M for the 127k, and 1 x M for the other samples. This solution was mixed with a five-fold excess (v/v) of matrix solution which was generally 2 x lo-' M in acetone and 1 p1 of this was placed on the probe and dried under ambient conditions.The mass spectrum of the pMMA 10k standard in t-3indoleacrylic acid acquired in the reflected mode is shown in Fig. 1. The molecular ions result from the cationization of the pMMA oligomers by fortuitous sodium and potassium with the periodicity of m/z 100 corresponding to the mass of the methyl methacrylate. The peak of the molecular ion distribution, M,, occurs at 5990 as opposed to the value of 9600 (determined by vapor-phase osmometry and intrinsic viscosity) claimed by the supplier. We suspected that selective cationization of the smaller oligomers could play a role here since the binding efficiency of polymers for alkali metal ions varies with the size of the polymer and nature of the ion for polyacrylates' and other polyelectrol...

The analysis of large molecules by matrix-assisted laser desorption time-of-flight mass spectrometry (MALD/TOF) is established as a powerful technique for the determination of molecular mass.',' The analysis of biopolymers such as prot e i n~,~-~ oligonucleotide^,^-^ and polysaccharideslO*'l by MALD/TOF provides molecular mass information faster and with more accuracy than previously available techniques such as polyacrylamide gel electrophoresis." Another class of large molecules for which this technique should provide important information is synthetic organic polymers. MALD/TOF has several advantages over conventional polymer molecular mass techniques such as gel permeation chromatography and vis-~0 m e t r y . I~ It provides absolute molecular masses as opposed to relative values, the entire distribution is determined instead of an average value for the molecular mass and molecular composition information is obtained since the molecular masses of single oligomers are measured. Previous results on polygly~ols~*'~ have been encouraging. Here we show results demonstrating the power of MALD/TOF for the determination of molecular mass distribution, polymer composition, and end group for selected water-soluble polymers.Measurements were performed using a Bruker REFLEXTM MALD/TOF mass spectrometer. This time-of-flight instrument is fitted with a reflectron and dual microchannel plate detector for high resolution analysis and with a postacceleration detector in the linear mode for lower resolution molecular mass distribution measurements. Samples were prepared in a sinapinic acid matrix at a mole ratio of to with a total loading of polymer around 20 to 100 pico-moles (this is based on the manufacturer's value for the average molecular mass). Ions were formed by laser desorption at 337 nm (N2 laser, 3 ns pulse width, lo7 to lo* 0.2 mm2 spot) and accelerated with 10-33 kV. The negative ions were detected in all cases, and ions less than m/z 1000 were removed with a pulsed deflector. The poly(acry1ic acid) sample (weight-average molecular mass," M, = 3000 Da) is supplied from Polysciences, Inc. and poly(styrene sulfonate) (peak-average molecular mass,I5 M, = 200000 Da) is from Polymer Laboratories. The polymers were supplied as the neutralized sodium salts of the acids. Since salts have been shown to have a deleterious effect on MALD ionization,6 the polymers were prepared in the acid form by ion-exchange with a column of Amberlite IRN-77 (Rohm and Haas Co.) ion-exchange resin followed by freeze-drying. The MALD/TOF mass spectrum of poly(styrene sulfonic acid) is shown in Fig.