RationaleChemical mass shifts in quadrupolar ion traps have been studied previously but only for a limited number of analytes and mass ranges. Here, mass shifts of cluster ions, commonly used as calibrants, and other analytes are qualitatively evaluated on the Bruker amaZon spherical ion trap (QIT) and the Finnigan LXQ linear ion trap (LIT). To extend the mass range from previous experiments m/z up to 4000 are investigated. MethodsChemical mass shifts of CsI, Y(HCOO)(3), and NaCF3COO cluster ions, CF3COO-, Na+, and Cs+ adduct ions, protonated commercial calibration solutions and peptides, and deprotonated peptides were investigated on the Bruker amaZon speed QIT and some of these were also investigated on the Finnigan LXQ LIT. ResultsOn both instruments, peak distortions and mass shifts toward lower m/z became apparent as m/z approached 1000. To some extent, the issues were more severe at slower scans. Peak distortions included loss of resolution, tailing, or fronting and were different between the amaZon QIT and the LXQ LIT. The noncluster and nonadduct ions analyzed showed no obvious mass shifts or peak distortions under the same analysis conditions. ConclusionsAs expected, the ion traps investigated here showed mass shift and peak distortion issues, and such issues persisted at m/z up to 4000 on both instruments. Peak distortions were different between the amaZon QIT and the LXQ LIT, and were not always visible despite mass shifts. Both mass shifts and peak distortions make cluster ions and some adduct ions unsuitable for ion trap calibration.

There is need for well-defined lignin macromolecules for research related to their use in biomaterial and biochemical applications. Lignin biorefining efforts are therefore under investigation to meet these needs. The detailed knowledge of the molecular structure of the native lignin and of the biorefinery lignins is essential for understanding the extraction mechanisms as well as chemical properties of the molecules. The objective of this work was to study the reactivity of lignin during a cyclic organosolv extraction process adopting physical protection strategies. As references, synthetic lignins obtained by mimicking the chemistry of lignin polymerization were used. State-of-the-art nuclear magnetic resonance (NMR) analysis, a powerful tool for the elucidation of lignin inter-unit linkages and functionalities, is complemented with matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF MS), to gain insights into linkage sequences and structural populations. The study unraveled interesting fundamental aspects on lignin polymerization processes, such as identifications of molecular populations with high degrees of structural homogeneity and the emergence of branching points in lignin structure. Furthermore, a previously proposed intramolecular condensation reaction is substantiated and new insights into the selectivity of this reaction are introduced and supported by density functional theory (DFT) calculations, where the important role of intramolecular π-π stacking is emphasized. The combined NMR and MALDI-TOF MS analytical approach, together with computational modeling, is important for deeper fundamental lignin studies and will be further exploited.

Calibration is vital for accurate m/z measurements. This review informs on calibration and possible calibrants for ESI-MS, highlighting analytical considerations important for high accuracy and precision. External calibration is often easier to perform, but the best accuracies are obtained with internal calibration, especially with calibrants close to and bracketing the analyte(s). Clusters of CsI allow the highest m/z calibration points, but other clusters give more closely spaced signals (clusters are unsuitable for ion trap calibration). Polymers such as PEG and PPG give closely spaced signals over a limited mass range, but suffer from memory effects. Commercial calibration solutions are easy to use and good for general calibration, but may be expensive and hard to modify. Proteins, peptides, small organic molecules, and some other calibrants offer high customization and may be preferred for more specific analyses, often for analysis of similar molecules. Because both accuracy and precision need to be known before measuring unknowns, the instrument should be evaluated. To avoid interferences, such as overlapping signals, it is important to keep the instrument and solutions free from contamination. Overlapping signals that cause shifts can, but may not, result in peak asymmetry, and smoothing can further obscure partially resolved interferences. Although increased resolution may resolve interfering signals, increased resolution can lead to lower sensitivity, potentially leading to loss of precision and accuracy due to poor ion statistics. Low intensity can to some extent be compensated by averaging more spectra. Trapping instruments suffer from space charge effects, so for the best results the number of ions needs to be limited but still high enough for sufficient ion statistics, and matching between calibration and analysis.

Rationale A calibration solution for mass spectrometry needs to cover the range of interest with intense and sufficiently narrowly spaced peaks. Limited options fulfilling this may lead to compromises between performance and ease of use. SpheriCal(R)-ESI was designed to combine high calibration performance for electrospray ionization (ESI) mass spectrometric analysis of peptides in positive mode with quick and easy use. Methods The developed calibration solution was tested using three mass spectrometers: two ion traps and one tandem quadrupole. The m/z errors of SpheriCal(R)-ESI itself and of a tryptic digest of cytochrome C were measured after calibration. The results were compared with those achieved with ESI Tuning Mix. The memory effects of the dendrimers, and contamination from Na+ in the calibration solution, were evaluated. Results SpheriCal(R)-ESI showed good shelf life as powder and was quickly reconstituted for use. Achieving intense and stable signals was straightforward. The accuracies and precisions were as expected for the instruments. SpheriCal(R)-ESI was more precise and at least as accurate as ESI Tuning Mix. The memory effects and Na+ contamination were found to be negligible in typical peptide solvents. In addition, the dendrimers showed predictable dissociations with product ions common to collision-induced dissociation in both ion trap and tandem quadrupole mass spectrometers. Conclusions SpheriCal(R)-ESI provided easily accessible calibration by showing intense signals at low infusion rates and at source settings equal or similar to those used in peptide analysis. Nine calibration points in the range of interest gave precise and accurate results. Memory effects and contamination were negligible even without rinsing.

 Mass spectrometry (MS) is an important analytical tool. Its most importantadvantages are sensitivity, resolution, and accurate and precise m/zmeasurements. To get the best results from an instrument, the user musthave knowledge of its limitation and requirements.The sensitivity of matrix assisted laser desorption/ionization (MALDI)-MSis dependent on the surface concentration of the sample. In Paper I, ahighly hydrophobic surface coating was developed and applied to MALDItarget plates. This allowed applied sample droplets to dry down ontosmaller areas. Compared to untreated and commercial concentrationplates, the surface treated plates gave on average twice the signal to noisefor peptide analysis.Signal suppression and signal overlap in MS can obscure analyte signals.In Paper II, an automated system for coupling capillary electrophoresis toMALDI-MS was developed. Separation of butterfly spermatophoreproteins into 32 fractions revealed some otherwise not detected oroverlapping MS signals.Calibration is vital for accurate MS measurements. In Paper III, adendrimer-based calibration solution for electrospray ionization (ESI)-MSwas developed, intended for peptide analysis. With a larger number ofsignals in the m/z range of interest, precision, and probably accuracy,improved compared to a commercial calibration solution.In Paper IV, calibration options for ESI-MS were reviewed. Factorsaffecting accuracy and precision, in general and for different instruments,were summarized, and a comprehensive list of calibrants was compiled.Paper V further investigated accuracy and precision in MS, specificallyregarding chemical mass shifts in ion traps. It was shown that clusters andsome adduct ions would be unsuitable for calibration of ion traps, and thatlinear ion traps showed mass shifts similar to spherical ion traps. 

 Masspektrometri (MS) är ett viktigt analytiskt verktyg. Dess viktigastefördelar är känslighet, upplösning, och exakta och precisa m/z mätvärden.För att få bästa resultat från ett instrument måste användaren känna tilldess begränsningar och krav.Känsligheten för matrisassisterad laser desorbtion/jonisering (MALDI)-MS är beroende av ytkoncentrationen av provet. I Paper I utvecklades enmycket hydrofob ytbehandling som applicerades på MALDI-provplattor.Det gjorde att provdroppar torkade på en mindre yta. Jämfört medobehandlade och kommersiella koncentrationsplattor gav ytbehandladeprovplattor i genomsnitt två gånger högre signal-till-brus vid peptidanalys.Signalkonkurrens och överlapp i MS kan dölja provsignaler. I Paper IIutvecklades ett automatiserat system för att koppla kapillärelektrofores tillMALDI-MS. Separation av fjärilsspermatoforproteiner i 32 fraktionervisade några annars odetekterade eller överlappande MS-signaler.Kalibrering är avgörande för exakta MS-mätningar. I Paper III utveckladesen dendrimerbaserad kalibrerlösning för elektrospray-jonisering (ESI)-MS, ämnad för peptidanalys. Med fler signaler i m/z-området av intresseförbättrades precision, och antagligen exakthet, jämfört med enkommersiell kalibrerlösning.I Paper IV utfördes en litteraturstudie av kalibreringsalternativ för ESIMS. Faktorer som påverkar exakthet och precision, i allmänhet och förolika instrument, sammanfattades, och en omfattande lista överkalibranter sammanställdes.I Paper V undersöktes exakthet och precision närmare i MS, specifikt medavseende på kemiska masskift i jonfällor. Det påvisades att kluster- ochadduktjoner vore olämpliga som kalibranter i jonfällor, och att linjärajonfällor gav masskift som liknade dem i sfäriska jonfällor. 

Osteopontin is an osteoblast-secreted protein with an aspartic acid-rich, highly phosphorylated, and glycosylated structure. Osteopontin can easily bind to integrins, tumor cells, extracellular matrix and calcium, and is related to bone diseases, various cancers, inflammation etc. Here, DEAE-Cibacron blue 3GA was used to extract recombinant osteopontin from human plasma, and to deplete abundant plasma proteins with an antibody-free method. Using selected buffer systems, osteopontin and human serum albumin could be bound to DEAE-Cibacron blue 3GA, while immunoglobulin G was excluded. The bound osteopontin could then be separated from albumin by using different sequential elution buffers. By this method, 1 μg/mL recombinant osteopontin could be separated from the major part of the most abundant proteins in human plasma. After trypsin digestion, the extracted osteopontin could be successfully detected and identified by MALDI-TOF MS/MS using the m/z 1854.898 peptide and its fragments.

A method for off-line CE-MALDI-TOF-MS and MS2, and on-target digestion under a fluorocarbon lid was developed and applied for the analysis of proteins in the spermatophore of the butterfly Pieris napi. Fractionation revealed many peptides otherwise not detected or resolved. Automated fractionation was performed with an in-lab developed robotic system, and automated on-target tryptic digestion under a fluorocarbon lid was demonstrated with the same system. Fractionation onto a pre-structured MALDI-concentration plate facilitated aligned deposition of trypsin and MALDI-matrix with the deposited sample, also under the fluorocarbon lid. Some indications of indigenous proteolysis of spermatophore proteins were seen, and searching MS2 spectra suggested three tentative sequence homologies to P. rapae. The study demonstrates the functionality of the lab-made robot. Detailed manufacturing instructions and code are provided. The feasibility of automated on-target digestion under a fluorocarbon lid, and the usefulness of a structured concentration plate in CE-MALDI fractionation was shown. Further, it constitutes a preliminary study of P. napi spermatophore proteins.

We report the modular formulation of ciprofloxacin-based pure theranostic nanodrugs that display enhanced antibacterial activities, as well as aggregation-induced emission (AIE) enhancement that was successfully used to image bacteria. The drug derivatives, consisting of ciprofloxacin, a perfluoroaryl ring, and a phenyl ring linked by an amidine bond, were efficiently synthesized by a straightforward protocol from a perfluoroaryl azide, ciprofloxacin, and an aldehyde in acetone at room temperature. These compounds are propeller-shaped, and upon precipitation into water, readily assembled into stable nanoaggregates that transformed ciprofloxacin derivatives into AIE-active luminogens. The nanoaggregates displayed increased luminescence and were successfully used to image bacteria. In addition, these nanodrugs showed enhanced antibacterial activities, lowering the minimum inhibitory concentration (MIC) by more than one order of magnitude against both sensitive and resistant Escherichia coli. The study represents a strategy in the design and development of pure theranostic nanodrugs for combating drug-resistant bacterial infections.

Here we present a method to manufacture peptide-concentrating MALDI-plates with alkyl ketene dimer (AKD) as a new superhydrophobic coating. The fabrication of the hydrophobic plates included application of AKD by airbrush, and negative contact printing to generate the concentration sites. Deposited sample droplets were contained within the prestructured sites, and self-adjusted onto the site if slightly misplaced. No AKD contamination was observed, and the plates could easily be cleaned and regenerated. The S/N values for four model peptides was about twice as high compared with a standard steel plate and a commercial concentration plate.

A method for off-line CE‑MALDI‑TOF-MS and MS², and on-target digestion under a fluorocarbon lid was developed and applied for the analysis of proteins in the spermatophore of the butterfly Pieris napi. Fractionation revealed many peptides otherwise not detected or resolved. Automated fractionation was performed with an in-lab developed robotic system, and automated on-target tryptic digestion under a fluorocarbon lid was demonstrated with the same system. Fractionation onto a pre-structured MALDI-concentration plate facilitated aligned deposition of trypsin and MALDI-matrix with the deposited sample, also under the fluorocarbon lid. Some indications of indigenous proteolysis of spermatophore proteins were seen, and searching MS² spectra suggested three tentative sequence homologies to P. rapae. The study demonstrates the functionality of the lab-made robot. Detailed manufacturing instructions and code are provided. The feasibility of automated on-target digestion under a fluorocarbon lid, and the usefulness of a structured concentration plate in CE-MALDI fractionation was shown. Further, it constitutes a preliminary study of P. napi spermatophore proteins.