Research method
Mass Spectrometry Proteomics
Mass-spectrometry proteomics identifies and, when labelled or quantified, counts peptides from a protein mixture after proteolytic digestion. Outputs range from a subcellular inventory (who is on this membrane face) to site maps of modifications (ubiquitin, lysine crotonylation) to subunit stoichiometry of a purified complex. An identified peptide is not a phenotype, and genome papers in this list are not proteomic experiments.
Cell biologists use MS when fractionation or antibodies cannot resolve a compartment or a modification site. It answers 'which proteins or modified lysines are in this sample?' The main limitation is that enrichment is not functional proof, and several lexicon hits here are genomes or transcriptomes that never ran a mass spectrometer on peptides.
Evidence
What the evidence shows
Drawn from 7 studies in this library. Each finding starts with a plain-language takeaway, then the denser detail. Supports means evidence for a finding; Challenges means evidence against a stated position; Qualifies marks scope with a short note on each study’s contribution. Challenged positions are labeled — they are not findings.
APEX2 proximity labelling in living cells defined cytosol-facing proteomes of the outer mitochondrial membrane and the ER membrane: expected functional classes plus many additions (22 OMM proteins; 72 ERM proteins), beyond classical fractionation. The inventory is not a full functional validation of every newly enriched protein.
Purified caveolar coats analysed by quantitative composition showed caveolins and cavins as one complex excluding EHD2/pacsin 2, with Cavin 1 a core component and evidence for trimers. MS-backed stoichiometry is a biochemical model, not a dynamics movie in every cell type.
Rab5 monoubiquitination sites were mapped at K116, K140 and K165; chemically monoubiquitinated Rab5 then showed that K140/K165 downregulate the endocytic pathway via disrupted effectors and nucleotide conversion, while K116 has little localisation effect. Site-resolved MS (or MS-informed chemistry) here is a modification map, not an identification of the endogenous E3 ligase.
In rice, MS plus ChIP-seq mapped histone lysine butyrylation and crotonylation: many Kcr sites on H3/H4, with Kbu/Kcr resembling acetylation distribution and changing with metabolic stress. Occupancy dynamics are not proof that every site is required for a named stress phenotype.
Three papers are false-positive lexicon hits for proteomics. Naegleria fowleri work assembled an ~30 Mb AT-rich genome and listed candidate pathogenicity genes; Aspergillus comparative genomics reported ~29–36 Mb genomes with 9,113–13,553 genes (~20% Aspergillaceae-specific; section Nigri ~1,800 unique genes); Plasmodium falciparum IDC transcriptome was hourly expression across a ~48-hour blood-stage cycle. Those are DNA or RNA catalogues, not peptide spectra.
- Pathogenicity factors in Naegleria fowleri
- Aspergillus genomic diversity
- How does malaria’s blood-stage transcriptome unfold?
Study Role Design N Population Outcome Pathogenicity factors in Naegleria fowleri Supports Computational / modellingDe novo genome sequencing plus proteomics to nominate N. fowleri pathogenicity factors ~29.6 Mb assembled nuclear genome (diploid estimate ~66 Mb); >500 million reads — genome resource study Naegleria fowleri (compared with non-pathogenic N. gruberi) Candidate pathogenicity gene sets from genome/proteome comparison Aspergillus genomic diversity Supports Computational / modellingComparative genomics of Aspergillus genomes in MycoCosm Multi-genome Aspergillus comparison (≈29–36 Mb; 9,113–13,553 genes) — genome count not a single primary N in stored summary Aspergillus / Aspergillaceae genomes Genome size, gene content, and clade-specific gene expansions How does malaria’s blood-stage transcriptome unfold? Supports Animal / in-vitroHourly IDC transcriptome of P. falciparum across the ~48-hour erythrocytic cycle Time-series parasite culture transcriptome — no single sample N Plasmodium falciparum during the intraerythrocytic developmental cycle Stage-ordered periodic gene expression across the IDC
Open questions
Tensions and limits
Some items are genuine disagreements on the same question. Others mark different assays, populations, or outcomes — limits on how far one study travels — not a forced fight between papers.
Genuine MS designs still disagree about what is being counted. APEX2 inventories membrane-face neighbours in living cells (22 OMM; 72 ERM); caveolar work quantifies a purified coat; Rab5 work maps three ubiquitin lysines; rice work maps histone acylations under stress. A 'proteomics paper' can be spatial, stoichiometric, or epigenetic, and those outputs are not interchangeable.
- Mapping the faces of mitochondria and ER
- What proteins build the caveolar coat?
- How does monoubiquitin turn down Rab5?
- Histone acylations respond to plant stress
Study Role Design N Population Outcome Mapping the faces of mitochondria and ER Supports Animal / in-vitroAPEX2 proximity labeling of cytosol-facing OMM and ER membranes in living HEK 293T cells SILAC proteomic maps with two replicates per membrane — cell-resource study HEK 293T cells expressing OMM/ERM APEX2 fusions Proteomes of cytosol-facing outer mitochondrial and ER membranes What proteins build the caveolar coat? Supports Animal / in-vitroBiochemical purification and ultrastructure of caveolar coat proteins Structural/biochemical coat stoichiometry study — no single sample N Caveolar coat complexes (caveolins/cavins) Molecular composition and ultrastructure of the caveolar coat How does monoubiquitin turn down Rab5? Supports Animal / in-vitroMapped Rab5 monoubiquitination sites and tested chemically monoubiquitinated Rab5 function Cell-biochemistry of Rab5 ubiquitin sites K116/K140/K165 — no single sample N Cultured cells expressing Rab5 variants Site-specific monoubiquitination downregulating Rab5 endocytic signaling Histone acylations respond to plant stress Supports Animal / in-vitroMS and ChIP-seq mapping of histone Kbu/Kcr in rice vs H3K9ac under metabolic stress Plant chromatin proteomics/ChIP-seq study — no single primary analytic N in stored text Rice (and related plants) under stress conditions Distribution and stress dynamics of histone butyrylation/crotonylation Naegleria and Aspergillus genomes and the Plasmodium IDC transcriptome should not be cited as mass-spectrometry evidence at all. Candidate pathogenicity gene lists and periodic RNA profiles are sequence resources that still need protein-level or infection-model tests.
- Pathogenicity factors in Naegleria fowleri
- Aspergillus genomic diversity
- How does malaria’s blood-stage transcriptome unfold?
Study Role Design N Population Outcome Pathogenicity factors in Naegleria fowleri Supports Computational / modellingDe novo genome sequencing plus proteomics to nominate N. fowleri pathogenicity factors ~29.6 Mb assembled nuclear genome (diploid estimate ~66 Mb); >500 million reads — genome resource study Naegleria fowleri (compared with non-pathogenic N. gruberi) Candidate pathogenicity gene sets from genome/proteome comparison Aspergillus genomic diversity Supports Computational / modellingComparative genomics of Aspergillus genomes in MycoCosm Multi-genome Aspergillus comparison (≈29–36 Mb; 9,113–13,553 genes) — genome count not a single primary N in stored summary Aspergillus / Aspergillaceae genomes Genome size, gene content, and clade-specific gene expansions How does malaria’s blood-stage transcriptome unfold? Supports Animal / in-vitroHourly IDC transcriptome of P. falciparum across the ~48-hour erythrocytic cycle Time-series parasite culture transcriptome — no single sample N Plasmodium falciparum during the intraerythrocytic developmental cycle Stage-ordered periodic gene expression across the IDC
Common misconceptions
If APEX2 enriches a protein on the OMM or ERM, that protein's function on that face is known.
The maps capture expected classes and add 22 OMM and 72 ERM proteins beyond classical fractionation. Enrichment is a location hypothesis, not functional validation of each new protein.
Detecting ubiquitin on Rab5 is enough to say the GTPase is off.
K140 and K165 modifications downregulate endocytosis; K116 has little localisation effect. Site-specific chemistry, not a bulk ubiquitin spectral count, made that distinction.
A pathogenicity-factor 'proteome' of Naegleria was measured because the paper is tagged for mass spectrometry.
The study sequenced and assembled an ~30 Mb AT-rich genome and nominated candidate genes. Those candidates still need functional validation in infection models and are not peptide identifications.
Exam-style questions
Short-answer questions that ask you to explain or compare, not recall.
Why can APEX2 list proteins that classical mitochondrial fractionation misses, and what can it still not claim?
Proximity biotinylation in living cells labels cytosol-facing OMM and ERM neighbours (22 and 72 proteins beyond expected classes). It does not prove each enriched protein's function or rule out labelling of transient visitors.
Caveolin–cavin coats exclude EHD2/pacsin 2. How is that a different MS question from mapping rice H3/H4 crotonylation sites?
Coat work is subunit stoichiometry of a purified complex (Cavin 1 core, trimers). Rice work is a modification atlas that resembles acetylation and changes with metabolic stress. One counts protein copies in a particle; the other counts modified lysines on histones.
Chemically monoubiquitinated Rab5 at K140/K165 versus K116 gave different endocytic outcomes. Why was mapping sites necessary before that experiment?
Without site maps (K116, K140, K165), a researcher would not know which lysines to modify. The chemistry tests those mapped sites; it still does not identify the endogenous E3 ligase.
Plasmodium IDC expression is highly periodic over ~48 hours. Why is that a poor example of mass-spectrometry proteomics?
The dataset is an hourly transcriptome from merozoite invasion through schizont stages, not a peptide inventory. Periodicity of mRNA is not a protein-level MS result and does not itself deliver a drug.
The studies
7 studies in this library bear on Mass Spectrometry Proteomics, ordered by citations.
- How does malaria’s blood-stage transcriptome unfold?
Plasmodium falciparum’s 48-hour intraerythrocytic cycle was profiled hourly, revealing tightly staged gene-expression programs after RBC invasion.
- Aspergillus genomic diversity
Comparative genomics shows Aspergillus species share genome size but harbor large lineage-specific gene sets.
- Mapping the faces of mitochondria and ER
APEX2 proximity biotinylation yields high-quality proteomic maps of cytosol-facing outer mitochondrial and ER membranes in living human cells.
- What proteins build the caveolar coat?
Caveolins and cavins purify as a caveolar coat complex with Cavin 1 as a core trimeric component.
- Histone acylations respond to plant stress
Rice histone butyrylation and crotonylation mark active chromatin and shift under starvation/submergence.
- Pathogenicity factors in Naegleria fowleri
Whole-genome analysis of N. fowleri highlights candidate pathogenicity factors for brain infection.
- How does monoubiquitin turn down Rab5?
Rab5 is monoubiquitinated at specific lysines; ubiquitin at K140/K165 disrupts effector binding and downregulates endocytic trafficking.
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