Ole Østergaard

Mass spectrometry (MS)-based proteomics aims to characterize comprehensive proteomes in a fast and reproducible manner. Here we present the narrow-window data-independent acquisition (nDIA) strategy consisting of high-resolution MS1 scans with parallel tandem MS (MS/MS) scans of ~200 Hz using 2-Th isolation windows, dissolving the differences between data-dependent and -independent methods. This is achieved by pairing a quadrupole Orbitrap mass spectrometer with the asymmetric track lossless (Astral) analyzer which provides >200-Hz MS/MS scanning speed, high resolving power and sensitivity, and low-ppm mass accuracy. The nDIA strategy enables profiling of >100 full yeast proteomes per day, or 48 human proteomes per day at the depth of ~10,000 human protein groups in half-an-hour or ~7,000 proteins in 5 min, representing 3× higher coverage compared with current state-of-the-art MS. Multi-shot acquisition of offline fractionated samples provides comprehensive coverage of human proteomes in ~3 h. High quantitative precision and accuracy are demonstrated in a three-species proteome mixture, quantifying 14,000+ protein groups in a single half-an-hour run.

Neutrophils are indispensable in the innate immune defense against invading microorganisms. Neutrophils contain SVs and several subsets of granules that are essential for their function. Proteins present in neutrophil SVs and granules are synthesized during terminal granulopoiesis in the bone marrow. The heterogeneity of granules, as determined by marker proteins characteristic of each granule subset, is thought to result from differences in the biosynthetic windows of major classes of granule proteins, a process referred to as targeting by timing. Qualitative proteomic analysis of neutrophil granules, SVs, and plasma membrane has been performed before. Here, we performed subcellular fractionation on freshly isolated human neutrophils by nitrogen cavitation and density centrifugation on a four-layer Percoll gradient. Granule subsets were pooled and subjected to SDS-PAGE, and gel pieces were in-gel-digested with trypsin. The resulting peptides were analyzed using LTQ Orbitrap XL tandem MS. A total of 1292 unique proteins were identified and grouped, according to the neutrophil fraction, in which they displayed maximal expression. In addition to various known neutrophil proteins, several uncharacterized proteins were found, as well as proteins not described previously in neutrophils. To study the correlation between mRNA expression in neutrophil precursors and the localization of their cognate proteins, the distribution of 126 identified proteins was compared with their mRNA expression profiles. The neutrophil subcellular proteome profiles presented here may be used as a database in combination with the mRNA array database to predict and test the presence and localization of proteins in neutrophil granules and membranes.

Papillon-Lefèvre syndrome (PLS) results from mutations that inactivate cysteine protease cathepsin C (CTSC), which processes a variety of serine proteases considered essential for antimicrobial defense. Despite serine protease-deficient immune cell populations, PLS patients do not exhibit marked immunodeficiency. Here, we characterized a 24-year-old woman who had suffered from severe juvenile periodontal disease, but was otherwise healthy, and identified a homozygous missense mutation in CTSC indicative of PLS. Proteome analysis of patient neutrophil granules revealed that several proteins that normally localize to azurophil granules, including the major serine proteases, elastase, cathepsin G, and proteinase 3, were absent. Accordingly, neutrophils from this patient were incapable of producing neutrophil extracellular traps (NETs) in response to ROS and were unable to process endogenous cathelicidin hCAP-18 into the antibacterial peptide LL-37 in response to ionomycin. In immature myeloid cells from patient bone marrow, biosynthesis of CTSC and neutrophil serine proteases appeared normal along with initial processing and sorting to cellular storage. In contrast, these proteins were completely absent in mature neutrophils, indicating that CTSC mutation promotes protease degradation in more mature hematopoietic subsets, but does not affect protease production in progenitor cells. Together, these data indicate CTSC protects serine proteases from degradation in mature immune cells and suggest that neutrophil serine proteases are dispensable for human immunoprotection.

OBJECTIVE: To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters. METHODS: Sixty-eight clinically well-characterized SLE patients, 38 healthy controls, 6 patients with systemic sclerosis (SSc), and 6 patients with rheumatoid arthritis (RA) were included. The numbers of annexin V-binding MPs displaying IgG, IgM, or C1q were enumerated by flow cytometry. MP protein levels were determined by mass spectrometry in clinically defined subsets of SLE patients and controls. The MP IgG load was determined by flow cytometric analysis of all samples from SLE patients and healthy controls. RESULTS: SLE patients had significantly increased total and relative numbers of IgG-positive MPs (P = 0.0004), with a much higher average IgG load per MP (P < 0.0001) than healthy controls. Quantitative mass spectrometry of purified MPs verified significantly increased IgG, IgM, and C1q levels in SLE patients. In RA and SSc patients, the average IgG load per MP was significantly lower than in SLE patients (P = 0.006 and P = 0.05, respectively). Also, the IgM load and C1q load per MP were significantly higher in SLE patients than in the control groups (P < 0.05), except for IgM in the RA group. IgG-positive MPs were significantly associated with the presence of anti-double-stranded DNA, anti-extractable nuclear antigen, and antihistone antibodies, with total IgG, and with decreased leukocyte counts. Average IgG load per MP was associated with lower concentrations of MPs, the presence of anti-C1q antibodies, and complement consumption. CONCLUSION: Our findings indicate that circulating cell-derived MPs in SLE patients carry increased loads of IgG, IgM, and C1q and that IgG MPs are associated with autoantibodies and complement activation. The findings link immunologic reactions on MPs with the etiology of SLE.

Germination of monocotyledonous plants involves activation and de novo synthesis of enzymes that degrade cell walls and starch and mobilize stored endosperm reserves for embryo growth. Two-dimensional (2-D) gel electrophoresis and mass spectrometry were applied to identify major water-soluble proteins in extracts of mature barley (Hordeum vulgare) seeds and to follow their fate during germination. About 1200 and 600 spots of pI 4-7 were detected on 2-D gels by silver staining and colloidal Coomassie Brilliant Blue staining, respectively. About 300 spots were selected for in-gel digestion followed by matrix-assisted laser desorption/ionization-mass spectrometry-peptide map fingerprint analysis. Database searches using measured peptide masses resulted in 198 identifications of 103 proteins in 177 spots. These include housekeeping enzymes, chaperones, defence proteins (including enzyme inhibitors), and proteins related to desiccation and oxidative stress. Sixty-four of the identifications were made using expressed sequence tags (ESTs). Numerous spots in the 2-D gel pattern changed during germination (micromalting) and an intensely stained area which contained large amounts of the serpin protein Z appeared centrally on the 2-D gel. Spots containing alpha-amylase also appeared. Identification of 22 spots after three days of germination represented 13 different database entries and 11 functions including hydrolytic enzymes, chaperones, housekeeping enzymes, and inhibitors.