Cell type- and brain region-resolved mouse brain proteome

In the paper

“Several studies have reported that quantities of messenger RNA and proteins often correlate poorly, emphasizing the importance of post-transcriptional processes that control protein synthesis and degradation11.”

What the sources say

• Supported

  In “Concentrations, production and turnover rates of mRNA and proteins at steady state,” the paper says mRNA and protein abundances correlate, but not strongly. In “Understanding steady-state mRNA and protein levels,” it identifies translation and protein degradation as major contributors to protein abundance beyond mRNA levels. (LLM, conf=0.94)

  cites [11] Vogel, C. & Marcotte, E.M. Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat. Rev. Genet. 13, 227–232 (2012). source

In the paper

“Previous studies of different human cancer cell lines have found that proteins tend to be ubiquitously expressed with differences in the level of expression of the proteins rather than in their absence or presence18–20.”

What the sources say

• Supported

  The Results report that 8,522 proteins—73% of those identified—were found in all eleven cell lines. The Discussion and Outlook adds that even ubiquitous proteins often vary severalfold in expression between cell lines, supporting the distinction between expression level and presence or absence. (LLM, conf=0.92)

  cites [18] Geiger, T., Wehner, A., Schaab, C., Cox, J. & Mann, M. Comparative proteomic analysis of eleven common cell lines reveals ubiquitous but varying expression of most proteins. Mol. Cell Proteomics 11, M111.014050 (2012). source
• Supported

  The Results and discussion (Antibody-based protein profiling) identifies three human cancer cell lines and reports that 83% of detected proteins were found in all three, while only 6% were exclusive to one. The Conclusions describe differences in expression levels across the lines, supporting the distinction between level and presence or absence. (LLM, conf=0.91)

  cites [19] Lundberg, E. et al. Defining the transcriptome and proteome in three functionally different human cell lines. Mol. Syst. Biol. 6, 450 (2010). source
• Partly supported

  The Results sections on 45 human cell lines and confocal microscopy report that most analyzed proteins were detected across cell lines, while 66% differed in expression level among the three lines examined by confocal microscopy. This supports the expression-level-versus-presence distinction, but our copy describes the lines as human cell lines without establishing the claim’s specific characterization of them as cancer cell lines. (LLM, conf=0.82)

  cites [20] Pontén, F. et al. A global view of protein expression in human cells, tissues, and organs. Mol. Syst. Biol. 5, 337 (2009). sourceExperimental — accuracy not yet measured

In the paper

“Astrocytes, in contrast, provide nutrients to the nervous tissue, maintain the extracellular ion balance, recycle neurotransmitters, shape synaptic circuits and regulate the blood-brain barrier24.”

What the sources say

• Partly supported

  “Astrocyte function during normal development” says astrocytes maintain extracellular homeostasis, and “Maturing astrocytes promote synaptogenesis in developing postnatal brains” describes their role in synapse formation. “Other possible developmental functions of astrocytes” says they regulate blood–brain barrier permeability. The passages do not establish the more specific claims that astrocytes provide nutrients or recycle neurotransmitters. (LLM, conf=0.87)

  cites [24] Molofsky, A.V. et al. Astrocytes and disease: a neurodevelopmental perspective. Genes Dev. 26, 891–907 (2012). sourceExperimental — accuracy not yet measured

In the paper

“These low abundance transcripts appear to be the result of rare stochastic transcription and are thought to often result in non-functional mRNAs28.”

What the sources say

• Supported

  In Results and discussion (page not provided), the paper concludes that low-expression genes likely reflect ‘leaky’ expression producing non-functional transcripts. It also says most have fewer than one transcript per cell on average and describes their expression as stochastic, matching the claim’s qualified wording. (LLM, conf=0.93)

  cites [28] Hebenstreit, D. et al. RNA sequencing reveals two major classes of gene expression levels in metazoan cells. Mol. Syst. Biol. 7, 497 (2011). source

In the paper

“As previously observed in ultra-deep proteome analysis, a relatively small number of proteins make up for a high proportion of the total protein mass15,34.”

What the sources say

• Supported

  The Results and discussion report that the 40 most abundant proteins comprised 25% of the measured HeLa proteome, while the most abundant 600 constituted 75% of its mass. This directly supports the cited observation that relatively few proteins account for a large share of protein mass. (LLM, conf=0.95)

  cites [15] Nagaraj, N. et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548 (2011). source
• Partly supported

  In “Coordinated Expression at mRNA and Protein Levels” (page not provided), the paper reports that the 20% most abundant proteins account for 81.3% of protein molecules in proliferating cells. This supports the unequal-abundance point, but the reported proportion is based on molecule counts, not total protein mass. (LLM, conf=0.85)

  cites [34] Marguerat, S. et al. Quantitative analysis of fission yeast transcriptomes and proteomes in proliferating and quiescent cells. Cell 151, 671–683 (2012). sourceExperimental — accuracy not yet measured

In the paper

“As observed previously, the abundance of the non-filtered data, expressed as reads per kilobase of exon per million fragments mapped (RPKM), revealed a bimodal distribution (Fig. 1e), where one third of the transcripts have abundances lower than 1 RPKM15,28.”

What the sources say

• Supported

  The Results and discussion section reports that the non-filtered RNA-Seq abundance distribution was bimodal and that about 33% of transcripts had signals below one FPKM. The source uses FPKM rather than RPKM, but it describes the same substantive finding for its single-end sequencing data. (LLM, conf=0.92)

  cites [15] Nagaraj, N. et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548 (2011). source
• Partly supported

  The Results and discussion describe two overlapping expression groups in RNA-seq data, but the RNA-seq distribution has a low-expression shoulder; the paper calls the microarray distribution clearly bimodal. Our copy does not report that one third of transcripts have abundances below 1 RPKM. Its figure of approximately 30% refers instead to genes with no detected reads at a sequencing depth of 25 million reads. (LLM, conf=0.86)

  cites [28] Hebenstreit, D. et al. RNA sequencing reveals two major classes of gene expression levels in metazoan cells. Mol. Syst. Biol. 7, 497 (2011). sourceExperimental — accuracy not yet measured

In the paper

“As the correlation between the proteomes and the corresponding transcriptomes was only 0.4 to 0.45,our analysis captured important differences between transcriptome and proteome30,31 (Fig. 2c).”

What the sources say

• Partly supported

  The section ‘Steady-state concentrations of protein molecules per mRNA and their correlates’ (page not provided) reports that several measurements have R² around 0.4 and that much of the variation in protein levels is not explained by mRNA levels. It does not give 0.4–0.45 as a general correlation coefficient: that figure is approximately a squared Pearson correlation, and the reported values vary across organisms. The citing authors’ statement about what their own analysis captured is their inference, not a finding attributed to this source. (LLM, conf=0.86)

  cites [30] de Sousa Abreu, R., Penalva, L.O., Marcotte, E.M. & Vogel, C. Global signatures of protein and mRNA expression levels. Mol. Biosyst. 5, 1512–1526 (2009). sourceExperimental — accuracy not yet measured
• ?Not checked — no full text of the sourcecites [31] Maier, T., Guell, M. & Serrano, L. Correlation of mRNA and protein in complex biological samples. FEBS Lett. 583, 3966–3973 (2009). source

In the paper

“Next, we transfected plasmids encoding for full-length Lsamp, Neurotrimin and Opcml tagged with EGFP into HEK293T cells, added soluble Fc fusion proteins and observed homo- and heterophilic interactions as noted previously41 (Supplementary Fig. 10).”

What the sources say

• Partly supported

  The section “Ntm forms homomeric complexes: potential role in promoting adhesion” reports homophilic binding between neurotrimin-expressing CHO cells and soluble Ntm-Fc. The paper also suggests heterophilic mechanisms in its neurite-outgrowth experiments, but does not establish the homo- and heterophilic interactions among Lsamp, Neurotrimin, and Opcml implied here. The HEK293T/EGFP experiment is the citing authors’ own work, not an experiment attributed to this source. (LLM, conf=0.87)

  cites [41] Gil, O.D., Zanazzi, G., Struyk, A.F. & Salzer, J.L. Neurotrimin mediates bifunctional effects on neurite outgrowth via homophilic and heterophilic interactions. J. Neurosci. 18, 9312–9325 (1998). sourceExperimental — accuracy not yet measured

In the paper

“Lsamp belongs to the IgLON family of adhesion molecules, which also comprises Ntm (neurotrimin), Opcml (opiod-binding cell adhesion molecule) and Negr1 (neuronal growth regulator1)37–39.”

What the sources say

• ?Not checked — no full text of the sourcecites [37] Pimenta, A.F. et al. The limbic system-associated membrane protein is an Ig superfamily member that mediates selective neuronal growth and axon targeting. Neuron 15, 287–297 (1995). source
• ?Not checked — no full text of the sourcecites [38] Schofield, P.R. et al. Molecular characterization of a new immunoglobulin superfamily protein with potential roles in opioid binding and cell contact. EMBO J. 8, 489–495 (1989). source
• Partly supported

  The Discussion (page unavailable in our copy) identifies LAMP, neurotrimin and OBCAM as members of a subfamily of Ig-like cell adhesion molecules, supporting the Lsamp, Ntm and Opcml portion of the claim. It does not identify Negr1 as a member or use the name IgLON. (LLM, conf=0.88)

  cites [39] Struyk, A.F. et al. Cloning of neurotrimin defines a new subfamily of differentially expressed neural cell adhesion molecules. J. Neurosci. 15, 2141–2156 (1995). sourceExperimental — accuracy not yet measured

In the paper

“Mutations in the gene are associated with a type of Stickler syndrome, a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects35.”

What the sources say

• Supported

  The Molecular genetics section reports COL11A1 mutations in families with the type 2 vitreous phenotype of Stickler syndrome. The Abstract describes Stickler syndrome’s ophthalmological and orofacial features, deafness, and joint disease, supporting the listed ocular, orofacial, auditory, and skeletal defects. Page numbers are not indicated for these passages. (LLM, conf=0.93)

  cites [35] Snead, M.P. & Yates, J.R. Clinical and molecular genetics of Stickler syndrome. J. Med. Genet. 36, 353–359 (1999). source

In the paper

“As positive controls, we used Necl1-Fc and Necl4-Fc42,43, which, as expected, bound to oligodendrocytes and axons, respectively.”

What the sources say

• Supported

  The Discussion reports that Necl1–Fc bound to oligodendrocytes in mixed cortical cultures. The Results report that Necl4–Fc robustly labeled axons of cultured DRG neurons. Page numbers were not supplied in our copy. (LLM, conf=0.92)

  cites [42] Spiegel, I. et al. A central role for Necl4 (SynCAM4) in Schwann cell-axon interaction and myelination. Nat. Neurosci. 10, 861–869 (2007). source
• Partly supported

  The Results section, “Necl proteins mediate adhesion to neurons and Schwann cells” (page not provided), reports Necl-4-Fc binding to DRG neurites, supporting the axon portion. It reports Necl-1-Fc binding to Schwann cells, not oligodendrocytes; the oligodendrocyte portion is not established by this paper. (LLM, conf=0.87)

  cites [43] Maurel, P. et al. Nectin-like proteins mediate axon Schwann cell interactions along the internode and are essential for myelination. J. Cell Biol. 178, 861–874 (2007). sourceExperimental — accuracy not yet measured

In the paper

“Ncam1 (Fig. 6b),a protein that negatively regulates myelination36.”

• Also stated in: § Discussion · plain

  “Ncam1, a protein that negatively regulates myelination36.”

What the sources say

• Partly supported

  The Discussion reports that PSA-NCAM acts as a negative signal for myelination, and the Results show increased myelination after masking or removing its PSA moieties. The citing sentence attributes this effect to Ncam1 generally, but the paper establishes it specifically for polysialylated NCAM, not NCAM regardless of its polysialylation state. (LLM, conf=0.87)

  cites [36] Charles, P. et al. Negative regulation of central nervous system myelination by polysialylated-neural cell adhesion molecule. Proc. Natl. Acad. Sci. USA 97, 7585–7590 (2000). sourceExperimental — accuracy not yet measured

In the paper

“The identified 17,634 transcripts from the RNA sequencing showed a bimodal distribution, where one-third of the transcripts were of low abundance, possibly as a result of stochastic transcription28.”

What the sources say

• Partly supported

  The Results and discussion describe two expression classes and an emerging low-expression peak after correcting for RNA-seq detection limits. They say the microarray distribution is clearly bimodal, however, while the uncorrected RNA-seq distribution has a low-expression shoulder. The passages do not substantiate 17,634 identified transcripts, a one-third low-abundance fraction, or stochastic transcription as the explanation for that fraction. (LLM, conf=0.87)

  cites [28] Hebenstreit, D. et al. RNA sequencing reveals two major classes of gene expression levels in metazoan cells. Mol. Syst. Biol. 7, 497 (2011). sourceExperimental — accuracy not yet measured

In the paper

“We observed differences in the proteome and transcriptome with Pearson correlation coefficients between 0.40 and 0.45, somewhat poorer than those previously reported for cell lines15, but in the range of recent comparisons of human tissues16.”

What the sources say

• Supported

  The Results and discussion report a proteome–transcriptome correlation of 0.6 in the HeLa cell line, supporting the cited comparison with a higher previously reported cell-line correlation. The 0.40–0.45 Pearson coefficients are the citing authors’ own observation, not a result attributed to this source; the source reports a Spearman correlation, so the metrics differ. (LLM, conf=0.87)

  cites [15] Nagaraj, N. et al. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 7, 548 (2011). source
• ?Not checked — no full text of the sourcecites [16] Wilhelm, M. et al. Mass-spectrometry-based draft of the human proteome. Nature 509, 582–587 (2014). source

In the paper

“We found that oligodendrocytes expressed the smallest number (341 proteins) of cell type-specific proteins, defined by the greater than tenfold fold enrichment criterion, and cortical neurons the largest (669 proteins).”

In the paper

“Between different cell types, correlations were much lower, from 0.48 to 0.84 for the proteome (Fig. 2a) and from 0.25 to 0.86 for the transcriptome (Supplementary Fig. 3).”

In the paper

“This corresponds to an average of 11,970 different proteins in each of these systems, of which 10,529 proteins were identified in all five cell types and brain (Fig. 1b,c).”

In the paper

“Similar to the comparison between the different cell types, GO-based enrichment analysis revealed that cell surface proteins were highly overrepresented in the group of the most abundant brain-versus liver-enriched proteins and four out of the top five GOCC terms were related to membranes (Supplementary Table 16).”

In the paper

“The cell type-specific proteins in microglia had functions in membrane trafficking (Rab32, Myo1f), lipid catabolism (Apobr, Lipa) and signaling at the cell surface (CD180, Dock2, Nckap1l,Gpnmb, Fermt3), whereas proteins with designated functions in actin-related processes (Cnn1, Fmn2, Nebl, Pdlim7, Synpo2) and cilia (Rsph1, Ccdc39) were enriched in astrocytes.”

In the paper

“In fact, in all four cell types, ∼ 30–40 of the most abundant proteins accounted for ∼25% of the total protein molecules, and some of these were cell type specific (for oligodendrocytes Mbp, Plp, Cnp1; for cortical neurons Atp1b1, Crmp1, Gpm6a; for astrocytes Gfap, Gstm1, Prdx6; and for microglia Gm5483; Fig. 3b).”

In the paper

“The large majority of proteins were expressed in all four cell types, whereas only about 15% of the quantified proteins could be considered to be cell type specific (>10-fold more abundant in one cell compared with all others; Supplementary Table 6).”

In the paper

“The top 50 cell type-specific proteins (the most enriched and abundant proteins) included many well-established cell type-specific markers, but, based on PubMed search, also a relatively large number of proteins that have previously not been studied in the context of the respective cell type (more than one-third of the 50 proteins in the various glial types).”

In the paper

“The 12,934 proteins identified in different CNS cell types correspond to 11,690 ENSEMBL genes, which represents ∽60% of all protein coding genes in the genome.”

In the paper

“Consistent with their hematopoietic origin, microglia were the most diverging cell type; and, as expected from their shared lineage, astrocytes and oligodendrocytes were closest to each other (Fig. 2b).”

In the paper

“As judged by principal component analysis (PCA) and hierarchical clustering, the optic nerve, the cerebellum and the brain stem were the most diverging brain regions, whereas the remaining seven regions clustered more tightly (Fig. 5a,b).”

In the paper

“Whereas Necl1-Fc, used as a positive control, promoted growth and spreading of oligodendrocytes, cells did not attach and grow on Lsamp-Fc coated coverslips (Fig. 8f,g).”

In the paper

“Using antibody staining, we confirmed the expression of Lsamp in oligodendrocytes and neurons, but not in astrocytes or microglia (Fig. 7e,f).”

In the paper

“In total, we identified 2,901 brain region-enriched proteins (>4-fold difference) and found that the thalamus expressed the smallest and the optic nerve the largest numbers of brain region-enriched proteins (154 and 1,179, respectively; Supplementary Tables 17 and 18).”

In the paper

“Immunofluorescence staining of cultured cells showed that Col11A1 can be used as a new marker for immature oligodendrocytes and immunohistochemistry of brain sections revealed a weak association of Col11A1 with white matter tracts (Supplementary Fig. 8).”

In the paper

“These analyses identified proteins of the extracellular matrix (Col11A1, Col1A1, Col1A2, Col4A1, Col3A1, Col5A2), of which Col11A1 was the most highly expressed and enriched protein.”

In the paper

“Notably, most family members were enriched in neurons and oligodendrocytes (except of Ntm, which was only enriched in neurons; Fig. 6c).”

In the paper

“Prph, Sncg and Serpinb1b were most abundant and enriched in the brain stem, Omp and S100a5 in the olfactory bulb, Ppp1r1b, Actn2 and Pde10a in the striatum, and Plcb4, Prkcd and Synpo2 in the thalamus (Fig. 5c).”

In the paper

“Microglia cells constitute the third class of glial cells with functions mainly related to immune response and maintaining brain homeostasis25.”

What the sources say

• ?Not checked — no full text of the sourcecites [25] Hanisch, U.K. & Kettenmann, H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat. Neurosci. 10, 1387–1394 (2007). source

In the paper

“Glia are not only abundant (constituting 50–90% of the cells in the human brain), but are also highly specialized toward cell type-selective tasks, and therefore ideally suited for these analyses21.”

What the sources say

• ?Not checked — no full text of the sourcecites [21] Barres, B.A. The mystery and magic of glia: a perspective on their roles in health and disease. Neuron 60, 430–440 (2008). source

In the paper

“Oligodendrocytes are primarily engaged in membrane synthesis, as their function is to insulate axons by enwrapping them with a multilamellar myelin sheath22,23.”

What the sources say

• ?Not checked — no full text of the sourcecites [22] Sherman, D.L. & Brophy, P.J. Mechanisms of axon ensheathment and myelin growth. Nat. Rev. Neurosci. 6, 683–690 (2005). source
• ?Not checked — no full text of the sourcecites [23] Aggarwal, S., Yurlova, L. & Simons, M. Central nervous system myelin: structure, synthesis and assembly. Trends Cell Biol. 21, 585–593 (2011). source

In the paper

“Between biological replicates of the same cell types, Pearson’s correlation coefficients were about 0.96, demonstrating excellent technical and biological reproducibility (Fig. 2a).”

In the paper

“The expression levels of cell type-specific markers for astrocytes (Aqp4, Gfap, Aldh1l1), oligodendrocytes (Cnp1, Mbp, Plp1), microglia (Iba1, Tlr2, Tlr7) and neuronal progenitors (Bcl11a, Bcl11b, Ebf3) showed that almost all of these markers were reproducibly enriched at least 16-fold, also confirming the quality of the proteome analysis (Supplementary Fig. 6a).”

In the paper

“The expression levels of well-described markers for astrocytes (Aquaporin 4, Gfap, Glast and Aldh1l1), oligodendrocytes (Cnp1, Sox10, Mbp, Mag, Mog, Plp1), cortical neurons (neurofilament, Snap25, synaptotagmin I, Gabra 2), cerebellar granule neurons (Synaptotagmin I, Snap25, Sv2b, Gprin1, L1cam) and microglia (Iba1, Tlr2, Tlr7, CD86) in our triplicate proteome analysis revealed that almost all of”

In the paper

“Together these analyses of our deep primary cell proteomes faithfully reproduce known and characteristic cellular functions as well as their markers, providing a solid foundation for their global characterization and for new discoveries.”

In the paper

“All four IgLONs are glycosylphosphati-dylinositol (GPI)-anchored glycoproteins with three Ig domains that have been implicated in cell adhesion and growth guidance in neurons, but not in glia.”

In the paper

“We filtered for 8,147 protein groups that were quantified in all replicates for at least one cell type or its differentiation stage.”

In the paper

“All statistical analyses were performed on at least three different biological replicates.”

In the paper

“our data and the rapidity with which it can now be generated, suggest that MS-based proteomics can be implemented more broadly into neuroscience to help resolve the full molecular complexity of the brain at the protein level, with cellular or even subcellular resolution.”

In the paper

“it tests and computes the difference of every protein annotation from the overall intensity distribution using the two-sided Wilcoxon-Mann-Whitney test where the multiple hypothesis testing is adjusted by applying a Benjamini-Hochberg FDR threshold of 0.02.”

In the paper

“Differentially expressed proteins were identified by an ANOVA based test at permutation-based FDR cutoff of 0.05.”

In the paper

“Protein identification required at least one unique or razor peptide per protein group.”

In the paper

“We performed two-sample welch t-tests to identify proteins with a significant differential expression in a cell type in comparison to others employing a 2% permutation-based FDR filter.”

In the paper

“only cultures of at least 95% purity were included in further experiments.”

In the paper

“Blinded quantification was performed by determining areas of the myelinated and unmyelinated axons.”

In the paper

“The sample size was similar or higher to those generally employed in the field.”

In the paper

“PLL coating (100 μg ml-1) was used as a positive control to verify the quality of the primary oligodendrocyte preparation.”

In the paper

“The replicates were grouped based on cell type and differentiation stage before applying an ANOVA test on the annotation matrix with a permutation-based FDR cutoff of 0.001 to identify annotation terms that are significantly different between at least two groups.”

In the paper

“We believe that these technological advances can be used broadly in the proteomic study of diverse and rare brain cell types.”

In the paper

“Apart from identifying a previously unknown negative regulator of myelination in specific fiber tracts of the CNS, these results exemplify the utility of our proteome resource.”

In the paper

“Commonly used filtering criteria forRNA-Seq reduced the number to 12,933, indicating that our proteome coverage is close to completion, at least in terms of detection of each functionally expressed gene.”

In the paper

“the required FDR was set to 1% at the peptide and protein level.”

In the paper

“Lsamp is known to mediate homophilic, as well as heterophilic, interactions with other members of the IgLON family48.”

What the sources say

• ?Not checked — no full text of the sourcecites [48] Gil, O.D. et al. Complementary expression and heterophilic interactions between IgLON family members neurotrimin and LAMP. J. Neurobiol. 51, 190–204 (2002). source

In the paper

“All animal experiments were performed according to the Lower Saxony State regulations for animal experimentation.”