Recommendations for reporting ion mobility mass spectrometry measurements

© 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc. Here we present a guide to ion mobility mass spectrometry experiments, which covers both linear and nonlinear methods: what is measured, how the measurements are done, and how to report the results, including the uncertainties of mobility and collision cross section values. The guide aims to clarify some possibly confusing concepts, and the reporting recommendations should help researchers, authors and reviewers to contribute comprehensive reports, so that the ion mobility data can be reused more confidently. Starting from the concept of the definition of the measurand, we emphasize that (i) mobility values (K0) depend intrinsically on ion structure, the nature of the bath gas, temperature, and E/N; (ii) ion mobility does not measure molecular surfaces directly, but collision cross section (CCS) values are derived from mobility values using a physical model; (iii) methods relying on calibration are empirical (and thus may provide method-dependent results) only if the gas nature, temperature or E/N cannot match those of the primary method. Our analysis highlights the urgency of a community effort toward establishing primary standards and reference materials for ion mobility, and provides recommendations to do so. © 2019 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc

Comparison of One- and Two-dimensional Liquid Chromatography Approaches in the Label-free Quantitative Analysis of <i>Methylocella silvestris</i>

The proteome of the bacterium <i>Methylocella silvestris</i> has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)–reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase–reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route

Analysis of MAHRP2 parasites.

(A)Western blots of parasites expressing the indicated MAHRP2 proteins are shown. Blots were probed with anti-mCherry or anti-GFP antibodies as indicated. (B)Western blot of parasites for comparison of expression levels of the indicated MAHRP2 proteins. The blots were probed with anti-mCherry (shown in red) and anti-plasmepsin V as a loading control (shown in green). (C) Immunofluorescence labelling of parasites expressing mCherry tagged MAHRP2:C-S11:DSLE. Intrinsic mCherry fluorescence of the proteins is shown in red. Labelling with anti-MAHRP1 is shown in green. (D) Immunofluorescence labelling of parasites expressing mCherry tagged MAHRP2:C-S11:DSLE and treated with Brefeldin A. Intrinsic mCherry fluorescence of the proteins is shown in red. Labelling with anti-plasmepsin V is shown in green. (E) Phase contrast and fluorescence images of parasites expressing mCherry tagged MAHRP2:C-S11:DSLE and treated with DMSO are shown. Scale bar: 2 μm. (TIF)</p

Fluorescence microscopy analysis of split-GFP expressing parasites.

(A-B) Cartoon representation of plasmepsin V with a C-terminal S11 tag (plasmepsinV:3xHA:C-S11), and phase contrast and green fluorescence images of parasites expressing GFP1-10 fragments together with plasmepsinV:3xHA:C-S11 are shown. (C-D) Images of parasites co-expressing cytoplasmic mCherry that has a C-terminal S11 tag with either ER-lumenal GFP1-10 or cytoplasmic GFP1-10 are shown. For increased clarity and comparison to figures in the main text, two brightness ranges are shown for each image, as indicated. For GFP and mCherry images in the main text brightness settings of 0–1000 and 0–800 were used, respectively. In the images shown here, 0–1000 and 0–800 are shown for GFP and mCherry, respectively, but a brightness setting of 0–4095 is also shown for both channels. (E-F) Images of parasites co-expressing ER-lumenal mCherry (ER-lumenal mCherry comprises the N-terminal signal peptide derived from PF3D7_0827900, mCherry, a C-terminal S11 tag, and a STREP tag, followed by an SDEL sequence) with either ER-lumenal GFP1-10 or cytoplasmic GFP1-10, are shown. (TIF)</p

Mass spectrometry methods.

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER-lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway.</div

ER-lumenal localisation of ER-retained Pf332.

(A-C) Phase contrast and fluorescence images of parasites expressing the indicated Pf332 proteins either alone or with the indicated GFP1-10 proteins. Scale bar: 2 μm.</p

Export of SBP1 is perturbed by a C-terminal ER-retention sequence.

(A-B) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. Scale bar: 2 μm. (C) The fraction of total mCherry fluorescence located within the parasite is shown for parasite lines expressing the indicated SBP1 and GFP1-10 proteins. Forty individual trophozoite stage parasites, from two independent experiments, were analysed for each parasite line. Data points for individual parasites, mean and standard deviation are shown. P-values were determined using a one-way ANOVA test, P < 0.0001 = ****. (D-E) For parasites expressing the indicated SBP1 proteins, the total mCherry fluorescence and total GFP fluorescence levels are plotted (for both channels this corresponds to the fluorescence in the infected red blood cell and the parasite). Forty individual trophozoite stage parasites, from two independent experiments, were analysed for each parasite line.</p

Transmembrane domain analysis.

During the blood stage of a malaria infection, malaria parasites export both soluble and membrane proteins into the erythrocytes in which they reside. Exported proteins are trafficked via the parasite endoplasmic reticulum and secretory pathway, before being exported across the parasitophorous vacuole membrane into the erythrocyte. Transport across the parasitophorous vacuole membrane requires protein unfolding, and in the case of membrane proteins, extraction from the parasite plasma membrane. We show that trafficking of the exported Plasmodium protein, Pf332, differs from that of canonical eukaryotic soluble-secreted and transmembrane proteins. Pf332 is initially ER-targeted by an internal hydrophobic sequence that unlike a signal peptide, is not proteolytically removed, and unlike a transmembrane segment, does not span the ER membrane. Rather, both termini of the hydrophobic sequence enter the ER-lumen and the ER-lumenal species is a productive intermediate for protein export. Furthermore, we show in intact cells, that two other exported membrane proteins, SBP1 and MAHRP2, assume a lumenal topology within the parasite secretory pathway. Although the addition of a C-terminal ER-retention sequence, recognised by the lumenal domain of the KDEL receptor, does not completely block export of SBP1 and MAHRP2, it does enhance their retention in the parasite ER. This indicates that a sub-population of each protein adopts an ER-lumenal state that is an intermediate in the export process. Overall, this suggests that although many exported proteins traverse the parasite secretory pathway as typical soluble or membrane proteins, some exported proteins that are ER-targeted by a transmembrane segment-like, internal, non-cleaved hydrophobic segment, do not integrate into the ER membrane, and form an ER-lumenal species that is a productive export intermediate. This represents a novel means, not seen in typical membrane proteins found in model systems, by which exported transmembrane-like proteins can be targeted and trafficked within the lumen of the secretory pathway.</div

Western blotting analysis of Pf332 expressing parasites.

(A) Western blots of parasites expressing the indicated proteins are shown. Blots were probed with anti-mCherry. (B) Western blots of parasites for comparison of expression levels of the indicated Pf332 proteins. The blots were probed with anti-mCherry (shown in red) and anti-plasmepsin V as a loading control (shown in green). (C) Western blots of parasites expressing the indicated proteins are shown. Blots were probed with anti-mCherry or anti-GFP as indicated. (TIF)</p

High contrast GFP images of MAHRP2-expressing parasites.

(A-C) Phase contrast and fluorescence images of parasites expressing the indicated proteins are shown. Proteins were expressed alone, co-expressed with ER-lumenal GFP1-10 or cytoplasmic GFP1-10, as indicated. Images are identical to those in the main text Fig 6 except that high contrast images of the GFP channel are shown. Contrast settings for GFP images are set at 0–200 to show weak GFP signal. Scale bar: 2 μm. (TIF)</p