Research will be limited to:
- Method: MALDI-TOF Mass Spectrometry
- Analyte: Proteins and/or Polymers
- Sample Preparation: Dried Droplet Technique
- Topic: Matrix Selection and Optimization
- Topic: Calibration

Strategy: I will investigate the specific matrix-to-analyte ratios and solvent systems optimized for the dried droplet technique, focusing on the distinct requirements for proteins versus synthetic polymers. I will also look for recent protocols regarding internal vs. external calibration standards and the impact of matrix selection on mass resolution and signal-to-noise ratios in MALDI-TOF MS.
Queries:
- MALDI-TOF matrix selection ratio proteins polymers dried droplet
- optimization of matrix-to-analyte ratio MALDI-TOF synthetic polymers
- MALDI-TOF calibration standards proteins polymers dried droplet method
- CHCA vs DHB vs SA matrix selection protein polymer MALDI-TOF
- dried droplet technique MALDI-TOF sample preparation protocol optimization

Executing 5 concurrent searches:
- MALDI-TOF matrix selection ratio proteins polymers dried droplet
- optimization of matrix-to-analyte ratio MALDI-TOF synthetic polymers
- MALDI-TOF calibration standards proteins polymers dried droplet method
- CHCA vs DHB vs SA matrix selection protein polymer MALDI-TOF
- dried droplet technique MALDI-TOF sample preparation protocol optimization

Found 8 papers. Key excerpts:

Disentangle a Complex MALDI TOF Mass Spectrum of Polyethylene Glycols into Three Separate Spectra via Selective Formation of Protonated Ions and Sodium or Potassium Adducts. — Xianwen Lou et al., 2022

Matrix assisted laser desorption/ionization
mass spectrometry (MALDI
MS) has not only revolutionized the analysis of proteins and peptides 1 , 2 but also provided a powerful and versatile method for the characterization
of synthetic polymers. 3 − 6 Synthetic polymers are macromolecules consisting of various numbers
of repeating units and can be complex mixtures with molecular weight
and end-group distributions. 7 One attractive
advantage of MALDI MS for polymer analysis is that ions recorded are
normally singly charged, which can greatly simplify the interpretation
of the MS results and facilitate the reliable reckoning of monomer
and end-group masses. 3 − 6 In spite of this, for some polymers with both high proton affinity
and high alkali metal ion affinity, such as the polyethylene glycol
(PEG) sample studied in this application note, protonated ions and
sodium and potassium adducts of the analytes can all be recorded concomitantly.
Sodium/potassium salts are even not required to be added intentionally
to the MALDI sample, because trace amounts of sodium/potassium impurities
ubiquitously present in the sample, glassware, solvents, and reagents
are usually sufficient to generate strong signals of the alkali metal
ion adducts. 8 The MALDI MS spectra for
these polymers can, therefore, still be very complicated and difficult
to interpret depending on the complexity of polymer distributions.
An ideal approach to tackle the complicated mass spectra would
be to distribute the ions into three separate mass spectra, with each
spectrum containing only protonated ions, sodium adducts, or potassium
adducts, respectively. For many oxygen-containing polymers with high
alkali metal ion affinity, directly adding a suitable salt of either
sodium or potassium in the MALDI sample was found to be a convenient
and effective way to form exclusively sodium or potassium adducts
of the polymer. 9 The high alkali metal
ion affinity, on the other hand, makes it difficult to generate protonated
polymer ions selectively without sodium/potassium adducts. Fortifying
the formation of protonated analyte ions by eliminating or reducing
sodium and potassium adducts is the subject of many studies on MALDI

applications, especially for biomolecules. 10 − 13 We report here a method to selectively
generate protonated ions for polyglycol samples.
It has been
widely recognized that the metal ion adducts are most
likely formed in the MALDI gas phase plume. 14 To eliminate the formation of the alkali metal ion adducts for a
polymer, therefore, a practical way is to inhibit the transfer of
Na + and K + and their related adduct ions into
the gas phase plume. In this application note, a PEG sample was selected
as the model polymer. α-Cyano-4-hydroxycinnamic acid (CHCA)
was used as the matrix, and octadecylamine (ODA) or a tetrabutylammonium
salt was used to suppress the release of Na + and K + and their related adducts. 15 , 16 The introduction
of co-matrixes has long been used to improve the quality of MALDI
measurements. 17 , 18 By depositing the polymer sample
on top of a preloaded layer of CHCA with a suitable co-matrix using
a modified thin-layer method, 19 selective
formation of protonated ions can be obtained for the polymer. The
aim of this work is to develop a method that can simplify MALDI MS
analysis of complex polymer samples. Evidently, by distributing various
ions into three mass spectra with each one containing only protonated
ions and sodium or potassium adducts, peak assignment can be greatly
simplified and be more reliable, especially in the analysis of a complex
polymer sample.
Three polymer samples were used in this study.
The samples of polyethylene glycol and polypropylene glycol with amino
end groups (PEG–NH 2 and PPG–NH 2 ) were obtained from Sigma-Aldrich (Zwijndrecht, The Netherlands),
and the PEG with hydroxy end groups (PEG–OH), from Polymer
Laboratories BV (Heerlen, The Netherlands). α-Cyano-4-hydroxycinnamic
acid (CHCA) was purchased from Fluka (Zwijndrecht, The Netherlands).
Octadecylamine, trihexylam

ine, ammonium fluoride, diammonium hydrogen
citrate, and tetrabutylammonium hexafluorophosphate were obtained
from Sigma-Aldrich.
Matrix solutions were freshly prepared
in THF or in a mixed solvent of water/acetonitrile (1/1 v/v with 0.1%
of trifluoracetic acid) at concentrations of approximately 20 mg/mL.
All of the sample solutions were also freshly prepared at concentrations
of about 1 mg/mL. Two sample deposition methods were employed in this
study, namely, the dried-droplet and modified thin-layer methods. 2 , 19 For the dried-droplet method, a sample solution and a matrix solution
were mixed in an Eppendorf tube. A 0.5 μL portion of the mixed
solution was pipetted onto a stainless steel MALDI plate and allowed
to dry. For the modified thin-layer method, a matrix solution and
a co-matrix solution were first mixed; then, 0.5 μL of the mixed
solution was deposited on the target plate and allowed to dry. After
that, a 0.5 μL aliquot of analyte solution in chloroform or
water was deposited on top of the first layer and allowed to dry.
Chloroform or water was chosen as the solvents for the polymers using
the modified thin-layer method with the aim to minimize the embedment
of analytes in the matrix crystals. 19
The MALDI TOF MS measurements were
performed with an Autoflex Speed (Bruker, Bremen, Germany) instrument.
The accelerating voltage was held at 19 kV and the delay time at 130
ns for all experiments. Mass spectra were acquired in the reflector
positive ion mode by summing spectra from 500 random laser shots at
an acquisition rate of 100 Hz.
A MALDI TOF mass spectrum of
a PEG sample obtained by using CHCA
matrix and the conventional dried-droplet sample deposition method
is shown in Figure 1 A. This PEG sample is supposed to be NH 2 C 3 H 6 –(OC 2 H 4 ) n –OC 3 H 6 NH 2 . PEGs are
known to be prone to form alkali metal ion adducts

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry — Molly E Payne et al., 2018

Introduction With improvements in living polymerization techniques, precision polymers with quantitatively functionalized end groups are increasingly available 1 . The concurrent development of azide-alkyne and thiolene click chemistries has enabled the nearly quantitative coupling of macromolecules to other moieties, providing access to a range of hybrid materials 2 3 4 . However, precise analytical techniques are required to characterize both the starting materials and products of these polymer conjugation reactions. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is a valuable soft ionization analytical technique for characterizing polymers because it can generate polymer ions in a single charge state with minimal fragmentation 5 6 . MALDI-TOF MS has major advantages over other conventional methods of polymer characterization because it can provide mass spectra with resolution of the individual n-mers within the polymer mass distribution. As a consequence, such mass spectra can provide precise information about the average molecular weight, repeat unit mass, and molecular weight dispersity 7 , which can in turn elucidate competing polymerization mechanisms such as chain transfer 8 . However, MALDI-TOF MS is particularly powerful at providing information about polymer end groups 9 10 , which can be used to confirm end group modifications 10 11 and other transformations 12 such as polymer cyclizations 11 13 . Equally important, the relatively small amount of analyte (sub-micrograms) required for mass spectrometric analysis makes this technique useful for characterization when only trace quantities of material are available. The MALDI-TOF MS analysis of polymers can be divided into four distinct steps: sample preparation, instrument calibration, spectral acquisition, and data analysis. Sample preparation is the most essential step for generating optimized MALDI-TOF mass spectra and occurs before the sample is introduced into the instrument 14 15 . The selection of an appropriate matrix with similar solubility parameters as the polymer analyte is critical to obtain high quality MALDI-TOF mass spectra and guidelines for matrix selection have been reported elsewhere 14 15 16 17 . A database of polymer MALDI "recipes" for sample preparation has also been published online 18 . For novel polymers, matrix selection can be approached by first understanding the solubility of the polymer and selecting a matrix with similar solubility parameters 14 19 . Polymers with high proton affinity can be protonated by most matrices 14 (

which frequently contain carboxylic acid groups), but for other polymers, a cationization agent is required 14 . Alkali ions adduct well with oxygen-containing species ( e.g . polyesters and polyethers), whereas unsaturated hydrocarbons ( e.g . polystyrene) adduct with transition metals such as silver and copper ions 14 19 . Because the polymer samples in this experiment contained oxygen atoms in the backbone, sodium or potassium trifluoroacetate (TFA) were used as the cationization agent. Once the matrix and cationization agents have been selected, the relative proportions of analyte, cation agent, and matrix must be carefully optimized to ensure high signal to noise. In this procedure, the parameters for sample preparation have already been optimized, however an empirical sample optimization procedure (step 1.4.1., Figure 1 ) that systematically varies the concentrations of the three components (analyte, matrix and cation) is effective for rapidly determining their optimal ratios. Data acquisition also requires the optimization of a number of parameters. The most important parameters include the positive or negative ion mode of the spectrometer, the instrument operation mode (linear versus reflector), the acceleration voltage, and the extraction delay time. Another way that resolution can be increased is through the utilization of "reflectron" mode 20 21 22 23 . Reflectron mode essentially doubles the flight path of the ions to the detector by reflecting the ions at the end of the flight tube back towards a detector near the source while refocusing ions with different momentums, and therefore increasing the resolution though decreasing signal strength. In addition, higher resolution spectra can be obtained by decreasing the laser power which minimizes the signal-to-noise ratio by decreasing the number and energy of collisions and therefore reducing the fragmentation and kinetic inhomogeneities 24 . By tuning all of these parameters, the ions can be focused to minimize the effect of any inhomogeneity in initial position or velocity that occurs during the laser desorption process. When the acquisition parameters are optimized, isotopic resolution can often be achieved for ions with masses in excess of 10,000 Da, though this is also dependent upon the length of the flight tube and the instrument design. Most organic compounds that contain at least one heteroatom are prone to complexing with alkali cations such as lithium, sodium, and potassium. Many of the alkali metals are monoisotopes or of

limited isotopes and therefore do not broaden the distribution. While the instrument parameters can be tuned to optimize data precision, data accuracy is only achieved with an appropriate calibration 11 . Proteins and peptides were originally used as calibrants due to their monodispersity and availability, but suffer from variable stability and the prevalence of impurities 25 . More cost-effective and stable alternatives have included inorganic clusters and polydisperse polymers 26 27 28 29 . Unfortunately, these alternatives feature disperse masses, which complicate mass assignments, as well as smaller masses overall, making them useful only for calibrations below 10,000 Da. To combat these issues, Grayson et al . 25 developed a dendrimer-based, polyester MS calibration system that is monodisperse, and boasts both broad matrix and solvent compatibility, shelf-life stability (> 8 years), and lower production cost. Based upon the strengths of this system, it was selected as the calibrant for these experiments. There are two main types of calibration: internal and external 30 . When calibrating externally, a standard with masses that bracket that of the analyte are placed on the MALDI target plate in a different sample position than the analyte to generate a separate mass spectrum from which a calibration file can be generated. On the other hand, increased accuracy can often be achieved with an internal calibration, which involves mixing the calibrant with the analyte to obtain a hybrid spectrum with both calibrant and analyte signals. In the procedure described below, an external calibration was implemented. After proper calibration of the mass scale, accurate analyte mass data can be acquired. To ensure the most accurate calibration, it is important that the data acquisition occurs soon after the calibration. Finally, once the optimized, calibrated data sets were acquired, and the data were analyzed to provide structural information about the polymer samples. The spacing between n-mers within the polymer distribution can provide accurate measurement of the repeating unit mass. The number average molecular weight (M n ) and other mass distribution calculations ( e.g. , M w (weight average molecular weight) and Đ (dispersity)) can also be determined from the signal distribution in the mass spectra (step 4.2 for calculations). Perhaps most uniquely, in the case of homopolymers, the sum of the end group masses can be confirmed by determining the offset of the polymer distribution with respect to the mass of the repeating units alone. The information-

Importance of the Matrix and the Matrix/Sample Ratio in MALDI-TOF-MS Analysis of Cathelicidins Obtained from Porcine Neutrophils — Anna Smolira et al., 2015

The MALDI method (matrix-assisted laser desorption ionization) in combination with a time of flight mass spectrometer (TOF MS) is an indispensable tool for the detection and identification of molecular masses of “heavy” biomolecules, which due to thermal instability can not be mass analyzed by the laser desorption (LD) method that is traditionally used to measure small masses [ MALDI TOF MS is the method that provides rapid determination of molecular masses. Its attribute is also high sensitivity and no need for prior purification of the samples before the measurement, which is especially important in studies of biological samples with for example proteins, oligosaccharides, lipids, or peptides contained in them [ One of the factors that influences MALDI TOF MS analyses both in terms of sensitivity and resolution is sample preparation. Thus, the major challenge for such studies is to identify its optimal procedures. This is of particular importance in the case of biological specimen contained in physiologic fluids where very often low amounts of an investigated material are available for experiment, and additionally, it is complex and not purified. Since the introduction of MALDI in 1988, different procedures in the subsequent stages of sample preparation were tested:
The aim of the presented paper is to show the relationship between the matrix/sample ratio used in the MALDI sample preparation process and the quality of the obtained mass spectra. The literature concerning this topic refers mainly to commercially available samples that are composed of one type of an analyte such as for example cytochrome c [ The MALDI TOF MS method was previously used by the authors for determination of the content of neutrophil extract [ The presented investigations were carried out using samples (portions) of lyophilisate from porcine neutrophils (see “ PF-2, PR-39, PG-1, PG-2, and PG-3 belong to antimicrobial peptides with a direct antimicrobial effect, as well as a wide range of activities on the immune system, which can be taken into account when introducing new antibiotic treatments [ The studies were devoted to the problem of optimization of the sample preparation process for the MALDI TOF MS measurement to get sensitivity of the investigated cathelicidin detection as high as it was possible. In particular, the authors focused on

selecting the optimal matrix and determination such ratio of a matrix and a sample solution (
MALDI-TOF Instrumentation The MALDI TOF MS analysis was performed on the time of flight mass spectrometer constructed by the author and co-workers in the Department of Molecular Physics, Institute of Physics, Maria Curie—Sklodowska University, Lublin, Poland (Fig.  The accelerating voltage between the sample holder and the grounded electrode is 17 kV. Ions are detected by the two channel plate detector (Hamamatsu, Photonics Deutschland GmbH, Herrsching am Ammersee, Germany) operating at the voltage of 2.3 kV. The signal from the detector is then sent to a 500 MHz (1 G sample/s) HP 54615B digital oscilloscope (Hewlett Packard, Warsaw, Poland) where all the data is collected as mass spectra. To enhance signal to noise ratio, each mass spectrum is averaged from 256 results obtained for consecutive laser shots. Next, the mass spectra are transferred to a PC for processing. Materials and Methods For studies of the matrix effect on the MALDI TOF MS measurement synthetic PR-39 (RRRPRPPYLP RPRPPPFFPP RLPPRIPPGF PPRFPPRFP) with purity > 95 % as confirmed by high-performance liquid chromatography and mass spectrometry (NOVAZYM POLAND s.c. Poznan Science &Technology Park, Poznan) was used. The matrices: α-cyano-4-hydroxycinnamic acid (CCA), 2,5-dyhydroxybenzoic acid (DHB), sinapinic acid (SA), nicotinic acid, benzoic acid, and succinic acid were purchased from Sigma-Aldrich. For the second part of investigations, all the cathelicidins (PR-39, PF-2, PG-1, PG-2, PG-3) were obtained from the porcine neutrophils crude extract in the process of crude extraction and gel filtration chromatography. Described method is dedicated for isolation of cationic antimicrobial peptides of low molecular mass. Successive steps of formation of a portion of cathelicidin lyophilisate, which was directly used

for the MALDI TOF MS measurement, are shown in Fig.  The amount of a sample in the portion of lyophilisate was about 20 μg. Matrix solutions were prepared by dissolving 0.01 g of the matrix in 1 ml of ACN (acetonitrile) and 0.1 % TFA acid (1:1,
Cathelicidins are encoded in the genome as prepropeptides with a classical N-terminal signal peptide (conserved signal sequence), propiece (cathelin), and C terminal highly variable peptide [ Choosing a Proper Matrix- Studies on Synthetic PR-39 In Fig.  Importance of the Matrix/Sample Ratio Studies on the impact of the matrix/sample ratio on the mass spectra obtained were carried out by using CCA as a matrix, which appeared to be the optimal for MALDI TOF MS measurement of investigated cathelicidins. In Fig.  As it can be seen, the intensity of individual ion mass peaks strongly depends on the sample preparation in the MALDI TOF MS measurement. Quantitatively, it is shown in Fig.  Analogous measurements to those for the PF-2 peptide were performed for cathelicidins PR-39 (4716 Da), PG-1 (1955,6 Da), PG-2 (2055,6 Da), and PG-3 (2154,5 Da). The mass spectra containing their ion mass peaks obtained for the matrix/sample ratios (
Nowadays, MALDI TOF MS became an indispensable tool in qualitative, and in recent years also, more and more quantitative research of large biomolecules such as peptides, proteins, oligosaccharides, lipids, and many others contained in complex biological samples. Its main advantages are as follows: softness due to a matrix used in the sample preparation process, high sensitivity and high tolerance of impurities, and non-volatile buffers present in the sample. Additionally, through combination with a time of flight mass spectrometer (TOF MS), time of analysis is short, range of the measured masses is very high, and a mass spectrum obtained is simple to analyze compared to other methods of detection (for example electrospray mass spectrometry). Despite its many advantages, a researcher must keep in mind that obtaining a good result in

Quantification in MALDI-TOF mass spectrometry of modified polymers. — Zuzana Walterová et al., 2011

Introduction

The resolution of MALDI-TOF mass spectrometry (MS) is so high that the molecular mass of individual species can be determined and thus the polymer modification, e.g., at the end groups, can be verified . Techniques such as delayed extraction and the reflectron mode increased the MALDI-TOF MS resolution so that signals corresponding to the individual polymerization degrees and end group modifications can be obtained at molecular mass well above 10,000 . On the other hand, the relation between the signal intensity and the content of a compound in the sample is rather weak due to poor reproducibility and composi-tional and molecular-mass discrimination . In spite of these random and systematic uncertainties, the MALDI-TOF MS quantification of biomacromolecules in biological tissue or fluids has been demonstrated viable. The response variability can be significantly decreased by the use of a suitable internal standard and, therefore, a reliable calibration curve, accounting for compositional and molecular-mass discrimination, can be generated [5,. The use of internal standard was also suggested for the quantification of synthetic polymers [12,13]. The generation of an internal-standard based calibration curve requires both the internal standard and the quantified compound to be available in a pure state. This can be a problem with synthetic polymers because that means a sample with identical molecular mass distribution (MMD) is available. On the other hand, the information sought with synthetic polymers is not usually the absolute concentration of a polymer in some solution; rather, it is the relative content of a particular polymer in some polymer mixture. An example is the determination of the content of a homopolymer in a copolymer or the content of a precursor polymer in a modified polymer after their synthesis. The latter case specifically deserves serious exploration because the precursor polymer is usually available and thus the internal-standard quantification is possible.
The pure modified polymer, however, will still not be available in most cases and consequently, the calibration curve will be impossible to determine. Fortunately, initial studies on the internal-standard quantification of synthetic polymers [12,13] found that the ratio of MS signal intensities of modified and precursor polymers (MS ratio) is proportional to the ratio of their concentrations in the sample (gravimetric ratio). Under such circumstances, it is not necessary to generate a calibration curve explicitly, and the method of standard addition can be used, i.e

., the correct gravimetric ratio can be obtained from the spectra of the analyzed sample and the sample with some precursor (analyte) added. The modified polymer is taken as an internal standard. The concept is developed here using poly(ethylene glycol) (PEG) and monomethyl poly(ethylene glycol) (MPEG) of similar MMD as a model system. First, the conclusions of previous studies are tested, namely (i) the correlation between the analyte and standard MS intensities and (ii) proportionality between the MS and gravimetric ratios. The first finding is corroborated but not the second one; therefore a new quantification procedure is developed and verified.

Experimental

Materials

Poly(ethylene glycol) monomethyl ether with nominal molecular mass of 2000 (MPEG), toluene, and anthracene-1,8,9-triol (dithranol) were obtained from Aldrich; poly(ethylene glycol) with nominal molecular mass of 2000 (PEG) from Fluka.

PEG/MPEG solutions preparation

Series of PEG/MPEG solutions were prepared from stock solutions (20 mg/mL) of PEG and MPEG in toluene. In the first series, 100 L of the PEG stock solution was mixed with 50, 100, 200, and 300 L of the MPEG solution, respectively and overall volume was adjusted to 400 L with toluene, if necessary. In the second series, the PEG and MPEG were swapped. In the third and forth series, mixtures of PEG/MPEG 1:2 and 2:1 were prepared at overall concentration of 20 mg/mL and diluted 1:1 with toluene three times.
The PEG and MPEG stock solutions used for testing the effect of the solvent and salt (i.e., cationization agent) were prepared in concentration of 10 mg/mL in dimethylformamide and mixed in volume ratios 1:2, 1:1 and 2:1.

MALDI-TOF mass spectrometry

The samples were prepared by the dried droplet method: solutions of PEG/MPEG mixtures and of a matrix (anthracene-1,8,9triol; 20 mg/mL) in toluene were mixed in the volume ratio 1:5, and

0.5-1 L of the mixture was deposited on the ground-steel target plate. MALDI TOF MS spectra were acquired with a Biflex III mass spectrometer (Bruker Daltonics) in the positive ion reflectron mode, using delayed extraction. The mass resolution achievable by the instrument was >12,000 for somatostatin and mass accuracy was better than 8 ppm for substance P using external calibration. The spectra were the sum of 300 shots with a N 2 laser emitting at 337 nm and consisted of peaks corresponding to PEG or MPEG adducts with a single Na + ion. (The spacing between adjacent isotopic peaks in all isotopic clusters was ~1 Da.) Peak areas were Fig. 1. Comparison of MALDI-TOF spectra of PEG and MPEG used in the quantification studies with selected peaks labeled to show the same regular peak spacing in both spectra due to the common monomer and the shift of the MPEG spectrum due to the modification of one end group. determined using the procedure SNAP of software FlexAnalysis (Bruker Daltonics), assuming PEG elemental composition.
The experiments testing the effect of a solvent and salt were carried out in a similar way, using dimethylformamide instead of toluene. In addition, sodium trifluoroacetate (NaCF 3 COO) was used as a cationization agent in the experiments testing the effect of salt; solutions of the sample, the matrix, and NaCF 3 COO were mixed in a volume ratio of 4:20:1.

Results and discussion

The model precursor/modified polymer pair consisted of poly(ethylene glycol) (PEG) and poly(ethylene glycol) monomethyl ether (MPEG) with similar molecular mass distributions (see Fig. 1). The quality and reproducibility of MALDI-TOF mass spectrum depends strongly on the sample-preparation method, including the matrix, ionization agent and solvent used. For that reason, we tested with PEG various deposition methods (dried droplet, thin layer, solventless); matrices (dithranol, dihydroxybenzoic acid, 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]- malononitrile); and solvents (THF,

Matrix normalized MALDI-TOF quantification of a fluorotelomer-based acrylate polymer. — Keegan Rankin et al., 2015

INTRODUCTION

Fluorinated polymers are the largest class of commercial fluorochemical products. Despite this fact, there is little known about their environmental fate and potential impact due to difficulties in developing analytical methods to directly measure changes in the formal polymer structure.
Fluorotelomer-based acrylate polymers (FTACPs) are a class of fluorinated polymers widely used as antiwetting and antistaining agents in the textile, upholstery, carpet, and paper industries. 1 Specifically, FTACPs are copolymers prepared from fluorotelomer acrylates (FTACs), hydrocarbon acrylates, and often other nonfluorinated monomers. 2,3 Similar to other fluorinated polymers, FTACPs benefit from improved repellency, lubricity, and chemical and thermal stability through the replacement of hydrogen with fluorine. 4 Consequently, the properties that make FTACPs ideal for industrial applications have also raised concern about their environment fate. Recent studies suggest that the degradation of FTACPs is likely an indirect source of the ubiquitous and persistent perfluoroalkyl carboxylates (PFCAs). 5,6 Because long-chain PFCAs (>7 perfluorinated carbons) have been demonstrated to accumulate in biota, 7,8 and the desired antiwetting and antistaining properties of the original FTACP formulation required fluorotelomer acrylates having >=8 perfluorinated carbons, 9-11 there is significant interest in directly assessing the environmental fate of FTACPs. Previous efforts have aimed at evaluating the degradation of FTACPs indirectly by measuring the transformation products (i.e., PFCAs) by high performance liquid chromatography tandem mass spectrometry (LC-MS/ MS). 12,13 However, PFCAs are also known transformation products of other fluorotelomer-based material such as FTACs and fluorotelomer alcohols (FTOHs), 14-20 which have been reported as residuals in the crude FTACP material at levels <5% (w/w). 21 Thus, indirect analysis of FTACPs degradation often requires the measurement of transformation products above a high background signal. Alternatively, degradation of FTACP itself could be monitored using a direct analysis method. Our group recently developed a qualitative matrixassisted laser desorption/ionization time-of-flight (MAL

DITOF) mass spectrometry method to investigate the degradation of FTACPs as a complementary method to conventional LC-MS/MS. 5 Although qualitative, the MALDI-TOF results clearly indicated alterations to the FTACP's chemical structure caused by microbial degradation. Besides this qualitative MALDI-TOF method, there does not appear to be another direct analysis methods for FTACPs.
MALDI-TOF is a technique often used to estimate the weight-average molecular weight (M w ), number-average molecular weight (M n ) and polydispersity index (PDI), as defined in the Supporting Information (SI), and provide structural characterization of synthetic polymers. 22-25 Despite having been introduced in the 1980s, 26,27 MALDI-TOF has remained primarily a qualitative analytical technique because of poor sample-to-sample reproducibility. Successfully obtaining a MALDI mass spectrum first requires a sample preparation that is compatible with the synthetic polymer properties to ensure intimate contact with the matrix; 28 considering the chemical diversity of synthetic polymers, this is not always trivial. Conventional solvent-based sample preparations, such as the dried droplet method, 29 rely on proper matrix and solvent selection to increase the likelihood of cocrystallization of the polymer and matrix upon solvent evaporation. To some degree, an appropriate matrix can often be selected with prior knowledge regarding the relative hydrophobicity and polarity of a polymer, 30,31 but may require tailoring of unique sample preparation for more problematic polymers. 32,33 The choice of a solvent is preferably a single or azeotropic system that allows the polymer, matrix, and cationization agent to be prepared together. 34-36 In addition, the rate of solvent evaporation is a contributing factor to polymer and matrix cocrystallization with faster evaporating solvents improving the sample homogeneity. 33,37 Modifications to the deposition method using thin-38,39 and seed-layered, 40 and electrospray 41-43 have been reported to further enhance sample homogeneity and improve the shot-to-shot reproducibility. However, even with these improvements, MALDI-TOF remains difficult to use for quantification with sufficient precision.
A number of studies have recently emerged reporting quantitative MALDI-TOF analysis of microcystins, 44 polymer additives, 45 saccharides, 46 peptides,

47,48 proteins 49,50 and synthetic polymers. 51-53 For synthetic polymers, use of an internal standard polymer is possible if it has similar chemical properties to the target polymer to ensure no discrimination during sample preparation. In addition, the mass spectrum of the internal standard polymer cannot overlap with that of the target polymer. The signal intensities or peak areas of the target polymer are then normalized to those of the internal standard polymer. The result minimizes sample-to-sample variability caused by differences in desorption, ionization and crystallization. Although studies have shown this to be an effective approach of quantifying synthetic polymers, 51-53 processing two overlaid polymer mass spectra can be rather tedious and time-consuming. This makes the work of Ahn et al. on the MALDI-TOF quantification of peptides using the matrix itself as an internal standard an appealing alternative. 54 The authors demonstrated that a linear calibration curve can be generated by taking the ratio of peptide to matrix signal intensities if the matrix suppression caused by the peptide concentration is <70%. If applicable for synthetic polymers, this approach would obviate the need for an internal standard polymer because the matrix itself would serve as an internal standard.
The aim of the present study was to develop a MALDI-TOF method using the matrix signal as an internal standard to directly quantify FTACPs. The model FTACP used in this study was poly(8:2 FTAC-co-HDA) copolymerized from 8:2 fluorotelomer acrylate (8:2 FTAC) and hexadecyl acrylate (HDA), with its chemical structure shown in Figure 1. Using a similar approach to Ahn et al., 54 the intensity of a matrix-cation cluster was used to normalize poly(8:2 FTAC-co-HDA) signal intensities to minimize the sample-to-sample variability. Calibration curves were generated using a series of poly(8:2 FTAC-co-HDA) standards, which allowed for reliable MALDITOF quantification whenever the same matrix solution was used to prepare both the poly(8:2 FTAC-co-HDA) standards and samples. To supplement our understanding of sample homogeneity, scanning electron microscopy (SEM) was used to provide surface distribution images following crystallization. Future application of this quantitative MALDI-

A sample preparation method for recovering suppressed analyte ions in MALDI TOF MS — Xianwen Lou et al., 2015

Introduction

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is an indispensable analytical tool for the characterization of a wide variety of compounds from small molecules to biosynthetic and synthetic polymers. MALDI and electrospray ionization (ESI) are the two most important 'soft' ionization techniques that allow for the sensitive detection of non-volatile and labile compounds with no or little fragmentation. Compared with ESI, one attractive feature of MALDI is that analytes are typically singly charged. [4,5] This feature makes the resulting mass spectra simple to interpret. Equipped with high-resolution mass analyzers, MALDI MS is, in principle, a very powerful technique for the direct analysis of multicomponent mixtures.
In real MALDI MS applications, however, the situation can be much more complicated. An essential requirement in the direct analysis of multicomponent mixtures is to obtain consistent signals simultaneously for all the analytes of interest. It is well known that, in MALDI MS, the ion intensity of a given analyte can be strongly influenced by co-existing components. For some mixtures, this influence is so severe that certain compounds can be completely suppressed or undetectable at all. An effective way to nullify this influence is to separate the mixture into individual analytes or fractionate into several analyte groups by chromatographic or electrophoretic methods prior to MALDI analysis. For many applications, off-line hyphenation of separation/fractionation with MALDI MS analysis is a useful combination. For applications such as rapid identification and validation of sample identity based on mass profile, direct MALDI MS analysis of mixtures is clearly advantageous in terms of the increased analysis speed and simplicity provided that the analyte suppression effect (ASE) of co-existing compounds can be eliminated or minimized.
ASE is a frequently observed MALDI phenomenon. It can be caused by the competitive desorption and/or ionization between co-existing components. The outcome of the competition is affected by many factors including the choice of matrix, the physicochemical properties of the analytes, the sample preparation method and the solvent and pH value of the MALDI solution. It can be envisaged that a bigger difference in desorption/ionization between analytes will lead to a stronger ASE. In

order to reduce the ASE, therefore, methods should be designed to weaken the competition for desorption and ionization. Regarding desorption, it has been reported that analytes can more easily be released into the gas phase from the matrix surface than from within matrix crystals. Incorporation of analytes into matrix crystals is not a prerequisite for MALDI MS and may be obstructive. With solvent-free methods, the embedment of analytes in matrix crystals can be minimized, and analytes are most likely in intimate contact with the matrix surface. Indeed, Wang and Fitzgerald reported that a solid-solid sample preparation protocol can significantly reduce the ASE of peptide mixtures. As for ionization, ions in MALDI can either be preformed in the condensed state or formed via charge transfer reactions in the gas phase. Because the ratio of ions to neutral species is only 10 A4 -10 A7 in the MALDI plume, [21,22] analytes in a multicomponent mixture must compete for the limited gas phase charges available for ionization. If possible, pre-charging the analytes may be a viable route to weaken the competition for ionization. Although pre-charged analytes require separation from counter ions, they will unlikely quench other ions of the same polarity in the gas phase because it would be thermodynamically unfavorable for ions to plunder extra charges in the MALDI plume. Based on this line of arguments, it seems that depositing (precharged) analytes on the surface of matrix could weaken the competition for desorption and ionization, and thus reduce the ASE in the analysis of multicomponent mixtures.
In this article, we report a modified thin-layer method to reduce the ASE in MALDI TOF MS. Three types of multicomponent mixtures of peptides, synthetic polymers and lipids were investigated. a-Cyano4-hydroxycinnamic acid (CHCA) was used as the matrix in this study because it is one of the most frequently used matrices in MALDI MS. Our results clearly indicate that analyte ion signals for the test samples, which were completely suppressed using the conventional dried-droplet method, could be satisfactorily recovered by depositing the analytes on the surface of matrix using our method.
Experimental a-Cyano-4-hydrocycinn

amic acid was purchased from Fluka (Zwijndrecht, The Netherlands), and tetrahydrofuran (THF, analytical reagent grade stabilized with butylated hydroxytoluene), chloroform (HPLC grade stabilized with amylene) and formic acid (HPLC grade) was purchased from Biosolve (Biosolve BV, Valkenswaard, The Netherlands). The following compounds, leucine enkephalin (LE), methionine enkephalin (ME), angiotensin II (AT), substance P, methoxypolyethylene glycol amine (MPEGA, average molecular weight 750 g/mol), glyceryl tridecanote (GTD), glyceryl trioleate (GTO), glyceryl trilinoleate (TGL) and 1,2dipalmitoyl-rac-glycero-3-phosphocholine (DPGPC) were all obtained from Sigma-Aldrich (Zwijndrecht, the Netherlands).
Gramicidin S was synthesized by Fmoc-solid-phase peptide synthesis using an automated solid-phase peptide synthesizer (Intavis MultiPep RSi, INTAVIS Bioanalytical Instruments AG, Koeln, Germany). Methoxypolyethylene glycol trimethyl ammonium (MPEGTMA) iodide was made by reacting MPEGA with iodomethane in chloroform. Matrix solutions were freshly prepared either in THF at a concentration of approximately 20 mg/ml or in a mixed solvent of water/acetonitrile (1 : 1 v/v) as a saturated solution. All the sample solutions were also freshly prepared. Unless noted otherwise, an equimolar peptide mixture solution of LE, ME and AT was first prepared in water, and a solution of substance P and gramicidin S (molar ratio of substance P/gramicidin S = 10:1) in water with 0.1% (v/v) formic acid. These solutions were then diluted two times either in acetonitrile for the conventional dried-droplet sample preparation method or in water for the modified thin-layer method. The PEG samples and the lipid samples were dissolved in chloroform.
For the dried-droplet sample preparation, the saturated

Dual roles of [CHCA + Na/K/Cs]+ as a cation adduct or a protonated salt for analyte ionization in matrix-assisted laser desorption/ionization mass spectrometry — Xianwen Lou et al., 2021

In matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS), matrix ions are generated by laser irradiation, and the secondary charge transfer reactions between these matrix ions and analyte molecules are essential for the ionization of analytes.1-5 α-Cyano-4-hydroxycinnamic acid (CHCA) is one of the most commonly used matrices due to its ability to efficiently generate analyte ions for various types of compounds. When CHCA is used as a matrix, protonated ions and alkali metal ion adducts are normally observed in the positive ion mode. Accurate interpretation and assignment of the various types of matrix ions are crucial to understand how MALDI ions are formed and how subsequent ionization of target analytes proceeds. Although [CHCA + Na/K]+ is usually assigned as sodiated/potassiated matrix molecules, we demonstrate that these ions are not simply alkali metal ion adducts. We show that they can be transformed into protonated ions of the corresponding matrix salts. The interconversion of these matrix ions has yet to be seriously interrogated thus far.

     Figure 1 shows a typical MALDI TOF MS spectrum of CHCA. The major peak at m/z 212.0 is [CHCA + Na]+, and is usually assigned as the sodium ion adduct of CHCA. Because of the ubiquitous presence of sodium impurities, addition of extra salts is generally not required for the formation of the alkali metal ion adduct. Most likely, [CHCA + Na]+ is formed in the gas phase by the adduction of Na+ to the matrix molecule, and, therefore, [CHCA + Na]+ is intuitively called the Na+ adduct of the matrix.2-4 However, by considering the molecular structure, it is also possible that this sodiated molecule can be transformed into a protonated ion of CHCA sodium salt (see Scheme 1).






         FIGURE 1Open in figure viewerPowerPoint

              A typical MALDI TOF MS spectrum of CHCA







              SCHEME 1Open in figure viewerPowerPoint

              Transformation between [(CHCA)Na]+ and [{CHCA − H + Na}H]+




     In Scheme 1 [{CHCA}Na]+ denotes the sodiated matrix molecule, and [{CHCA − H + Na}H]+ represents the protonated ion of CHCA sodium salt. Based on this reaction, [CHCA + Na]+ can swap between alkali metal ion adducts and protonated ions. This interchange reaction is extremely important to a full understanding of the secondary charge transfer reactions between matrix ions and analyte molecules in MALDI. For the ionization of analyte molecules, sodiated matrix molecules can provide Na+ while protonated ones can provide H+. We demonstrated that [CHCA + Na]+ can yield an alkali metal ion as [{CHCA}Na]+ or yield a proton as [{CHCA − H + Na}H

]+ to an analyte molecule depending on the properties of the analytes.

     The MALDI TOF MS measurements were performed with an Autoflex Speed instrument (Bruker, Bremen, Germany) equipped with a 355 nm Nd:YAG smartbeam laser with maximum repetition rate of 1000 Hz, capable of executing both linear and reflector modes. The accelerating voltage was held at 19 kV and the delay time at 130 ns for all experiments. Mass spectra were acquired in the reflector positive ion mode by summing spectra from 500 random laser shots at an acquisition rate of 100 Hz. Matrix solutions were freshly prepared in tetrahydrofuran (THF) at a concentration of approximately 20 mg/mL. Sodium trifluoroacetate (NaTFA), potassium trifluoroacetate (KTFA) and CsI3 were also dissolved in THF at approximately 20 mg/mL. Polyethylene glycol (PEG with OH and H end groups, average molecular weight 600) and didecylamine (DDA) were selected as the analytes for the alkali metal ion transfer and the proton transfer reactions, respectively.

     It is relatively straightforward to determine whether a matrix ion is a Na+ adduct. As a cation adduct, Na+ will be stripped from the matrix as compounds with higher Na+ affinity are introduced, resulting in suppression of the sodiated matrix molecule. PEGs are well-known to have a high Na+ affinity. Figure 2 shows a MALDI TOF MS spectrum of PEG-600 with the CHCA matrix. Indeed, high PEG peaks were observed at the expense of [CHCA + Na]+. The charge transfer reaction can be described by Scheme 2.

                    (2)Following this scheme, [CHCA + Na]+ is acting as a Na+ adduct. It can be seen in Figure 2 that the [CHCA +

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa — Luca Signor et al., 2013

Introduction

Structural biology relies on the production of high quality proteins 1 and therefore needs to be coupled with efficient and reliable techniques for protein analysis 2,3 . In our mass spectrometry (MS) laboratory within an institute of structural biology we need to confirm primary sequence of proteins, evaluate the presence of mutations and posttranslational modifications, the degradation of proteins, the sample homogeneity, and the quality of isotopic labelling (e.g. deuterated proteins for nuclear magnetic resonance studies 4 ). Since structural biologists use limited proteolysis to distinguish structurally rigid domains from flexible parts, we need to reliably characterize such truncated proteins using MS.
When biomolecules are analyzed by MS, two possible approaches are utilized to softly ionize such heavy and labile molecules. Electrospray ionization (ESI) ionizes molecules directly from the liquid phase 5 ; matrix-assisted laser desorption ionization (MALDI) requires that the biomolecules are co-crystallized with ultraviolet-absorbing organic molecules (i.e. matrix molecules) 6 . ESI time-of-flight (TOF) MS coupled to liquid chromatography has become a routine technique for the analysis of intact proteins, because it allows mass determination with high accuracy (<= 50 ppm). However, it is quite susceptible to the composition of the sample buffer (in particular to salts and detergents) and to contaminants (i.e. polymers) which are sometimes difficult to eliminate, causing the suppression of the analyte signal.
MALDI-TOF MS represents an effective alternative to ESI-MS because its performance is less affected by buffer components, detergents, and contaminants, and allows intact protein mass determination with sufficient accuracy (<= 500 ppm) for sequence validation. After protein digestion, MALDI-TOF MS can be also utilized to analyze the obtained peptides for further primary sequence confirmation by the so-called "peptide mass fingerprinting". , such method yields high sensitivity when a biomolecule mass is above 100 kDa. Remarkable analyses of intact proteins were carried out using home-made instruments between the end of the 1980's and the beginning of the 1990's . MALDI-TOF can be also used as a screening tool to evaluate the quality of protein samples because it requires less time for sample preparation and is less susceptible to interferences due to common impurities (e.g. salts).

After a first, quick evaluation by MALDI-MS, a sample can be further analyzed by ESI-TOF to determine its mass with higher accuracy. Furthermore, MALDI generates ions containing fewer charges than ESI and therefore acquiring and interpreting MALDI data is more straightforward. This allows students working in structural biology to analyze their recombinant proteins just after a brief training.
Two key factors influence the quality of the MALDI spectra: the matrix and the technique used for the matrix deposition (e.g. dried droplet 8 and thin layer 17,18 ). A single organic matrix [e.g. sinapinic acid (SA) or a-cyano-4-hydroxycinnamic acid (a-CHCA) 14,22,23 ] is often used for the MS examination of intact proteins and cross-linked protein complexes. Using a-CHCA, Chait group previously presented a detailed protocol for preparing an ultra thin layer prior to MALDI analysis of soluble and membrane proteins 14,15 . Recently, Gorka et al. illustrated the graphitebased target coating to improve the MALDI analysis of peptides and proteins using a-CHCA as matrix 24 .
Here, we present a simple protocol for the analysis of intact proteins by MALDI-TOF MS, utilizing a mixture of two matrices: 2,5-dihydroxybenzoic acid (DHB) and a-CHCA 25 . We systematically evaluated the performance of the DHB-CHCA mix compared to the SA and a-CHCA matrices, for the control of intact proteins. The matrix mixture allows a better resolution (i.e. the protein peaks are much sharper 26 . This is particularly useful for the molecular weight determination of proteins larger than 100 kDa.
Higher sensitivity is also reached using the DHB-CHCA mixture (0.5 pmoles of protein spotted on the MALDI target).
As we mentioned above, an important factor that should be considered when using MALDI instrument is the matrix deposition. Laugesen et al. proposed the use of DHB-CHCA mixture for the first time 25 , utilizing the dried droplet deposition. However, we observed better results (e.g. much higher sensitivity) when we utilized the thin layer method where the first layer is formed by the a-CHCA dissolved in acetone. The thin layer method 12

,27 implies the formation of a homogeneous substratum of matrix crystals on the MALDI target, which was described in a JoVE video previously 15 . Then, the sample is deposited on this substratum, and finally additional matrix is deposited (see below). In this article, we also illustrate how to deposit the sample on the MALDI target, but also how to clean the target 15 , to recrystallize, and prepare the matrices.
To conclude we aim to provide all the necessary information for analyzing intact proteins to scientists (in particular, structural biologists) who need to evaluate the quality of produced proteins in a rapid and simple way and who are not so familiar with MALDI-TOF MS. As predicted in mid-1990s 28 , MS has had an increased impact on biological research, as its accessibility to biologists has increased. We hope that the information we provided will be useful to make MALDI-TOF accessible to biologists and scientists who would like to start using mass spectrometry.

Protocol

Protein Sample Preparation: Buffer Exchange (Optional)

5-25 ml of micromolar concentrations of protein (1 to 20 mM) are necessary. Buffer exchange can be carried out using centrifugal ultrafiltration devices (e.g. Vivaspin, Sartorius) or microcentrifuge gel filtration columns (e.g., Micro Bio-Spin 6 chromatography columns, Bio-Rad) 29,30 . Buffer exchange steps can be repeated 2-3x . Detailed description of buffer exchange was previously presented 29,30 . For example, we utilize 20 mM tris(hydroxymethyl)aminomethane (i.e. Tris) pH 8 as final buffer.
Note: This step could be omitted in many cases. It should be carried out when a protein sample contains molecules or buffers that could strongly interfere with MS detection [e.g. glycerol; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, HEPES]. It is also useful to improve the quality of the spectra.

Recrystallization of Matrices in Order to Improve Their Purity (Optional)

1. Pour 10 ml of 40% ethanol (EtOH) into a Pyrex flask. 2. Add 600 mg of a matrix. 3. Using a water bath and a