Magnus Palmblad

Immunoglobulin G Glycopeptide Profiling by Matrix-Assisted Laser Desorption Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Immunoglobulin G (IgG) fragment crystallizable (Fc) glycosylation is essential for Fc-receptor-mediated activities. Changes in IgG Fc glycosylation have been found to be associated with various diseases. Here we describe a high-throughput IgG glycosylation profiling method. Sample preparation is performed in 96-well plate format: IgGs are purified from 2 microL of human plasma using immobilized protein A. IgGs are cleaved with trypsin, and the resulting glycopeptides are purified by reversed-phase or hydrophilic interaction solid-phase extraction. Glycopeptides are analyzed by intermediate pressure matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS). Notably, both dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) matrixes allowed the registration of sialylated as well as nonsialylated glycopeptides. Data were automatically processed, and IgG isotype-specific Fc glycosylation profiles were obtained. The entire method showed an interday variation below 10% for the six major glycoforms of both IgG1 and IgG2. The method was found suitable for isotype-specific high-throughput IgG glycosylation profiling from human plasma. As an example we successfully applied the method to profile the IgG glycosylation of 62 human samples.

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as (15)N. By utilising hydroponic media that contain (15)N inorganic salts as the sole nitrogen source, near to 100% (15)N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled (14)N- and (15)N-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of (14)N/(15)N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the (14)N and (15)N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct (14)N and (15)N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2).

Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface

Biological activities of immunoglobulin G such as effector functions via Fc receptor interactions are influenced by Fc-linked N-glycans. Here we describe a fast, robust and sensitive nano-LC-ESI-MS method for detailed subclass specific analysis of IgG Fc N-glycosylation. A sheath-flow ESI sprayer was used as a sensitive zero dead volume plug-and-play interface for online MS coupling, generating a very constant spray and ionization over the entire LC gradient. The propionic acid containing sheath-liquid effectively suppressed TFA gas-phase ion-pairing, enabling the use of TFA containing mobile phases. The fixed position of the sheath-flow ESI sprayer, far away from the glass capillary inlet, reduced MS contamination as compared to conventional nano-ESI. The method was found to be suitable for fast and detailed subclass specific IgG Fc N-glycosylation profiling in human plasma. The obtained subclass specific IgG Fc N-glycosylation profiles were processed automatically using in house developed software tools. For each of the IgG subclasses the 8 major glycoforms showed an interday analytical variation below 5%. The method was used to profile the IgG Fc N-glycosylation of 26 women at several time points during pregnancy and after delivery, revealing pregnancy-associated changes in IgG galactosylation, sialylation and incidence of bisecting N-acetylglucosamine.

Electron capture dissociation of substance P using a commercially available Fourier transform ion cyclotron resonance mass spectrometer

Electron capture dissociation of the peptide Substance P is reported for the first time, with an unmodified, commercially available Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. The fragmentation pattern is compared with that obtained with collisionally induced dissociation of the ions in the electrospray ion source, and note that electron capture dissociation gives a more easily interpreted spectrum, showing mainly C-fragments. With the exception of the proline residues, which require cleavage of two chemical bonds, we observe all C-fragmental we find the bias voltage of the electron gun not to be very critical.

Mass spectrometry in clinical proteomics - from the present to the future.

MS is an important analytical tool in clinical proteomics, primarily in the disease‐specific discovery, identification and characterisation of proteomic biomarkers and patterns. MS‐based proteomics is increasingly used in clinical validation and diagnostic method development. The latter departs from the typical application of MS‐based proteomics by exchanging some of the high performance of analysis for the throughput, robustness and simplicity required for clinical diagnostics. Although conventional MS‐based proteomics has become an important field in clinical applications, some of the most recent MS technologies have not yet been extensively applied in clinical proteomics. In this review, we will describe the current state of MS in clinical proteomics and look to the future of this field.

Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling.

We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing.

Automatic analysis of hydrogen/deuterium exchange mass spectra of peptides and proteins using calculations of isotopic distributions.

High mass-resolving power has been shown to be useful for studying the conformational dynamics of proteins by hydrogen/deuterium (H/D) exchange. A computer algorithm was developed that automatically identifies peptides and their extent of deuterium incorporation from H/D exchange mass spectra of enzymatic digests or fragment ions produced by collisionally induced dissociation (CID) or electron capture dissociation (ECD). The computer algorithm compares measured and calculated isotopic distributions and uses a fast calculation of isotopic distributions using the fast Fourier transform (FFT). The algorithm facilitates rapid and automated analysis of H/D exchange mass spectra suitable for high-throughput approaches to the study of peptide and protein structures. The algorithm also makes the identification independent on comparisons with undeuterated control samples. The applicability of the algorithm was demonstrated on simulated isotopic distributions as well as on experimental data, such as Fourier transform ion cyclotron resonance (FTICR) mass spectra of myoglobin peptic digests, and CID and ECD spectra of substance P.

Fibronectin is a serum biomarker for Duchenne muscular dystrophy

To identify and validate serum biomarkers for the progression of Duchenne muscular dystrophy (DMD) using a MS‐based bottom‐up pipeline.

A 9.4 T Fourier transform ion cyclotron resonance mass spectrometer: description and performance

9.4 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers (Bruker BioAPEX-94e) have been installed at the Division of Ion Physics, Uppsala University, and at the Department of Chemistry, University of Warwick. The BioAPEX-94e FT-ICR instrument is built around a high-field, superconducting magnet and a platform with easily interchangeable ion sources [matrix-assisted laser desorption/ionisation (MALDI), secondary ion mass spectrometry (SIMS), electrospray ionisation (ESI) and electron impact/chemical ionisation (EI/CI)]. In this paper a technical description of the instrument is given. Outstanding performance characteristics are demonstrated, notably clear resolution of C59N+ and C5813C2+ (mass difference 3.65 mDa) and mass measurement accuracy at the low ppm level. A wide range of applications in Warwick and Uppsala is described, demonstrating the versatility and high performance of the instrument.

Alignment of capillary electrophoresis–mass spectrometry datasets using accurate mass information

Capillary electrophoresis–mass spectrometry (CE–MS) is a powerful technique for the analysis of small soluble compounds in biological fluids. A major drawback of CE is the poor migration time reproducibility, which makes it difficult to combine data from different experiments and correctly assign compounds. A number of alignment algorithms have been developed but not all of them can cope with large and irregular time shifts between CE–MS runs. Here we present a genetic algorithm designed for alignment of CE–MS data using accurate mass information. The utility of the algorithm was demonstrated on real data, and the results were compared with one of the existing packages. The new algorithm showed a significant reduction of elution time variation in the aligned datasets. The importance of mass accuracy for the performance of the algorithm was also demonstrated by comparing alignments of datasets from a standard time-of-flight (TOF) instrument with those from the new ultrahigh resolution TOF maXis (Bruker Daltonics).