Sensitive Top-Down Proteomics Analysis of a Low Number of Mammalian Cells Using a Nanodroplet Sample Processing Platform
Open accessAbstract
Top-down proteomics is a powerful tool for characterizing genetic variations and post-translational modifications at intact protein level. However, one significant technical gap of top-down proteomics is the inability to analyze a low amount of biological samples, which limits its access to isolated rare cells, fine needle aspiration biopsies, and tissue substructures. Herein, we developed an ultrasensitive top-down platform by incorporating a microfluidic sample preparation system, termed nanoPOTS (nanodroplet processing in one pot for trace samples), into a top-down proteomic workflow. A unique combination of a nonionic detergent dodecyl-β-d-maltopyranoside (DDM) with urea as protein extraction buffer significantly improved both protein extraction efficiency and sample recovery. We hypothesize that the DDM detergent improves protein recovery by efficiently reducing nonspecific adsorption of intact proteins on container surfaces, while urea serves as a strong denaturant to disrupt noncovalent complexes and release intact proteins for downstream analysis. The nanoPOTS-based top-down platform reproducibly and quantitatively identified ∼170 to ∼620 proteoforms from ∼70 to ∼770 HeLa cells containing ∼10 to ∼115 ng of total protein. A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells. We anticipate the nanoPOTS top-down proteomics platform will be broadly applicable in biomedical research, particularly where clinical specimens are not available in amounts amenable to standard workflows.
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- Findingown data
Although high DDM concentration or other additives increased protein identifications, most of the additional proteoforms had masses below 10 kDa.
“Although the use of a high concentration of DDM or other additives all increased the protein identifications, most of the additionally identified proteoforms had a mass <10 k Da (Figure 3e).”
Quote in the paperSubstantial claim - Findingown data
About 60% of the identified proteoforms, on average, carried modifications when terminal truncations were excluded.
“Although only small numbers of cells were analyzed, our topdown proteomics data revealed average ~60% of identified proteoforms contained modifications (not counting terminal truncations) (Supporting Information -Excel Table S2-4).”
Quote in the paperNot a substantial claim - Findingown data
110 proteoforms and 61 proteins were common across samples with different cell counts.
“Among those, 110 proteoforms and 61 proteins were common in samples with different cell counts (Figure 4a-b).”
Quote in the paperNot a substantial claim - Findingown data
Urea produced the highest number of proteoforms with masses between 10 and 20 kDa compared with the other buffers.
“Compared to other buffers, urea also gave the highest number of proteoforms with mass of 10-20 kDa (Figure 3e).”
Quote in the paperSubstantial claim - Findingown data
No larger proteins appeared in the deconvoluted MS1 spectra.
“Larger proteins were not even observed in the deconvoluted MS 1 spectra (Figure S5).”
Quote in the paperSubstantial claim
- Findingown data
The most frequently observed modification was N-terminal acetylation.
“N-terminal acetylation was the most-common modification.”
Quote in the paperNot a substantial claim - Findingown data
The analysis also revealed several uncommon mass shifts, which were assigned to known PTMs in UniProt when possible.
“several uncommon mass shifts were also observed, and when possible, assigned to known PTMs in the UniProt database.”
Quote in the paperNot a substantial claim - Findingown data
One calmodulin proteoform carried N-terminal acetylation plus an extra mass shift of 42.047 Da close to its C-terminus.
“For example, a calmodulin proteoform was detected with N-terminal acetylation and an additional mass shift of 42.047 Da near the C-terminus (Figure 5a).”
Quote in the paperNot a substantial claim - Findingreasoning
PEG 20000 gave a marginal improvement in identifying proteoforms with masses between 10 and 20 kDa.
“On the other hand, PEG 20000 showed a marginal improvement for identification of proteoforms with mass 10-20 kDa possibly due to the reduced nonspecific binding (i.e.improved recovery of larger proteoforms).”
Quote in the paperNot a substantial claim - Findingown data
The nanoPOTS-based top-down platform reproducibly and quantitatively identified about 170 to 620 proteoforms from about 70 to 770 HeLa cells containing about 10 to 115 ng of total protein.
“The nanoPOTS-based top-down platform reproducibly and quantitatively identified ~170 to ~620 proteoforms from ~70 to ~770 HeLa cells containing ~10 to ~115 ng of total protein.”
Quote in the paperSubstantial claim - Findingown data
Among the four extraction buffers tested, the urea-containing buffer produced the highest numbers of proteoform and protein identifications.
“Comparison of the four extraction buffers (buffers 4-7 in Table 1) showed that the urea-containing buffer produced the highest number of proteoform and protein identifications (Figure 3a-d).”
Quote in the paperSubstantial claim - Findingown data
Simply increasing DDM concentration did not significantly improve identification of higher molecular mass species.
“Simply increasing DDM concentration did not show significant improvement for identification of higher molecular mass species.”
Quote in the paperSubstantial claim - Findingown data
Among the tested extraction conditions, the DDM-based buffer was the most effective for maximizing identifications from about 100 HeLa cells, based on the numbers of identified proteoforms and proteins.
“As shown in Figure 2, DDM-based extraction buffer was the most effective among all the conditions tested, as evidenced by the number of identified proteoforms (Figure 2a and 2c) and proteins (Figure 2b and 2d).”
Quote in the paperSubstantial claim - Findingown data
Several post-translational modifications, including acetylation, myristoylation, and iron binding, were identified using fewer than 800 cells.
“A variety of post-translational modifications including acetylation, myristoylation, and iron binding were identified using only less than 800 cells.”
Quote in the paperSubstantial claim - Findingown data
393 proteoforms and 90 proteins were exclusively identified in the 769±22 cell samples.
“A total of 393 proteoforms and 90 proteins were exclusively identified in the 769±22 cells.”
Quote in the paperSubstantial claim - Findingown data
At later points in the LC gradient, unresolved isotopic distributions dominated the mass spectra.
“By manually examining the data, we found that the mass spectra were dominated by unresolved isotopic distributions at the later stages of the LC gradient.”
Quote in the paperSubstantial claim - Findingown data
Proteoform MS peak intensities scale linearly with cell counts.
“In addition to proteoform identification, we also demonstrate the proteoform MS peak intensities scale linearly with cell counts, indicating the feasibility of label-free quantitation.”
Quote in the paperSubstantial claim - Findingown data
The water-only control gave a single protein identification, ubiquitin, in one of three replicates.
“In comparison, the control sample (water only) resulted in a single protein identification (i.e., ubiquitin) in 1 out of 3 replicates.”
Quote in the paperNot a substantial claim - Findingown data
The highest cell loading yielded the highest counts of protein and proteoform identifications.
“The highest number of cells not surprisingly yielded the highest counts of protein and proteoform identifications.”
Quote in the paperSubstantial claim - Findingown data
Charge state deconvolution with UniDec software detected larger proteoforms having molecular weights as high as 57 kDa.
“Using charge state deconvolution with UniDec software, 41 we were able to detect larger proteoforms with molecular weight up to 57 kDa (Figure S6).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [41] source
Not checked in the free run.
- Findingown data
Mass errors in the high-resolution spectra supported assigning the modification as trimethylation (42.047 Da) instead of acetylation (42.010 Da).
“Based on the mass errors in the high resolution mass spectra (Figure 5b-c), the modification can be confidently assigned as trimethylation (42.047Da), and not acetylation (42.010Da).”
Quote in the paperNot a substantial claim - Findingown data
Compared with commonly detected proteoforms, the unique proteoforms found in the 769±22 cell samples generally had low intensities.
“Compared to the commonly detected proteoforms, most of the unique proteoforms detected with 769±22 cells had relatively low intensities”
Quote in the paperNot a substantial claim - Findingown data
For the highest cell loading, more proteoforms and proteins were detected over the entire mass range below 30 kDa.
“We also observed more proteoforms and proteins were identified across the whole mass range (i.e.<30kDa) for highest cell loading, as indicated by the proteoform mass distributions in Figure 4c.”
Quote in the paperSubstantial claim - Findingown data
Despite increased total proteoform counts with higher cell loadings, few proteoforms above 20 kDa were detected.
“Despite the increase in the total number of proteoforms using higher cell loadings, there were still limited numbers of proteoforms detected above 20 kDa (Figure 4c).”
Quote in the paperSubstantial claim - Findingreasoning
Combining the nonionic detergent DDM with urea markedly improves protein extraction efficiency and sample recovery.
“We discovered that a unique combination of a nonionic detergent DDM with urea can significantly improve both protein extraction efficiency and sample recovery.”
Quote in the paperSubstantial claim - Findingown data
The majority of low-mass proteoforms correspond to less than half of the full-length sequences predicted from the genome.
“Our results indicate that most of the low mass proteoforms represent <50% of the full-length genome predicted sequences (Table S5)”
Quote in the paperSubstantial claim - Findingown data
An unusual mass shift of 52.9 Da near acidic residues led to identification of an iron-binding thymosin beta-4 proteoform.
“For example, an iron binding proteoform of thymosin beta-4 can be identified based on the unusual mass shift of 52.9 Da near acidic residues (Figure S9).”
Quote in the paperNot a substantial claim - Findingown data
For four chosen proteoforms, cell counts and proteoform intensities showed a linear relationship irrespective of mass or modification state.
“Regardless of their masses and modification states, a linear correlation can be observed between cell counts and proteoform intensities (Figure 6a-d).”
Quote in the paperSubstantial claim - Findingown data
The correlation held consistently for all proteins across a mass range extending to 23 kDa.
“The correlation was also consistent across all proteins spanning a mass range up to 23k (Figure 6f).”
Quote in the paperSubstantial claim - Findingown data
Overall, the correlation was strong, with a median R2 of 0.928.
“The correlation was high overall (median R 2 0.928).”
Quote in the paperSubstantial claim - Findingown data
The nanoPOTS top-down platform reproducibly and quantitatively identified about 170 to 620 proteoforms from roughly 70 to 770 HeLa cells with less than 120 ng total protein.
“We demonstrated the nanoPOTS top down platform can reproducibly and quantitatively identify ~170 to ~620 proteoforms from ~70 to ~770 HeLa cells containing <120 ng total proteins.”
Quote in the paperSubstantial claim - Interpretationown data
Revealing low-abundance protein and proteoform species required a larger number of cells.
“demonstrating more cells were required to reveal low abundant protein/proteoform species.”
Quote in the paperSubstantial claim - Premisecitation
In Fourier transform MS, larger proteins are harder to detect than smaller ones because their signals decay faster, owing to space-charge effects or collisions with background gas.
“This is not surprising, as detection of larger proteins in Fourier transform MS is more challenging due to faster signal decay than smaller proteins due to space-charge effects or collision with background gas.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [42] source
Not checked in the free run.
- Premisecitation
A previous study found that DDM, a mild detergent, only marginally improved protein solubility.
“A previous study indicated DDM, which is a mild detergent, only marginally improved protein solubility.24”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [24] source
Not checked in the free run.
- Interpretationown data
Label-free quantitation is feasible for this platform.
“In addition to proteoform identification, we also demonstrate the proteoform MS peak intensities scale linearly with cell counts, indicating the feasibility of label-free quantitation.”
Quote in the paperSubstantial claim - Interpretationreasoning
The improvement seen with urea is attributed to its chaotropic property, which disrupts noncovalent interactions and releases proteins from native complexes.
“The improvement could be attributed to the chaotropic property of urea, which efficiently disrupted noncovalent interactions and released proteins from native complexes.”
Quote in the paperSubstantial claim - Premisecitation
A knowledge-independent approach permits discovery of novel modifications.
“The knowledge-independent analysis, however, allows novel modifications to be discovered.51,52”
Quote in the paperNot a substantial claim0 of 2 sources checked - Interpretationown data
The combination of 0.1% DDM and 8 M urea was selected as the optimal buffer for subsequent experiments.
“Based on these results, we chose the combination of 0.1% DDM and 8 M urea as optimal buffer for subsequent experiments.”
Quote in the paperSubstantial claim - Premisecitation
High-kinetic-energy collisions during Orbitrap detection can cause fragmentation and marked signal decay.
“The collisions at high kinetic energies during Orbitrap detection can even induce fragmentation and significant signal decay.42”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [42] source
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- Interpretationown data
Scaling down sample processing with the nanoPOTS platform allows top-down proteomic analysis of small numbers of mammalian cells.
“In this study, we demonstrated ability to perform top-down proteomic analysis of small number of mammalian cells by scaling down the sample processing using the nanoPOTS platform.”
Quote in the paperSubstantial claim- Rests on an assumption: HeLa cells are representative of mammalian cells for the nanoPOTS top-down proteomics platform.
- Interpretationown data
Quantitation has a reasonably wide dynamic range, spanning at least about four orders of magnitude.
“The negligible dependence on proteoform intensity indicates the dynamic range of quantitation is reasonably high, at least across ~4 orders of magnitude (Figure 6e).”
Quote in the paperSubstantial claim - Interpretationreasoning
Degradation during prolonged sample preparation or storage before LC-MS analysis probably produced some of these low-mass proteoforms.
“some of which likely originated from degradation during lengthy sample preparation or storage prior to LC-MS analysis.”
Quote in the paperSubstantial claim - Interpretationown data
Label-free quantitation is a potential application of the nanoPOTS top-down platform.
“The high correlation of proteoform intensity with the amount of input material indicates that the nanoPOTS top-down platform could be used for label-free quantitation.”
Quote in the paperSubstantial claim
- May combine two claims. 21
- Classified as probably not a substantial claim. 16
May combine two claims.
Findingown dataIn samples with 74±7, 217±79, and 769±22 HeLa cells (n=3), the numbers of identified proteoforms were 174, 297, and 620, corresponding to 86, 120, and 198 proteins, respectively.
“As shown in Figure 4a, a total number of 174, 297, and 620 proteoforms (corresponding to 86, 120, and 198 proteins) were identified using 74±7, 217±79, and 769±22 HeLa cells (n=3), respectively”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataIn the urea-containing buffer, 32 of 207 unique proteoforms were ribosomal proteins, whereas in the other three buffers only 12 of 361 unique proteoforms were ribosomal proteins.
“Interestingly, among the 207 unique proteoforms detected in the urea containing buffer, 32 were ribosomal proteins.In contrast, among the 361 unique proteoforms detected in all the other three buffers tested, only 12 were ribosomal proteins (Supporting Information -Excel Table S1).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataDDM yielded an average of 96 proteoforms, whereas ProteaseMax and Invitrosol yielded averages of 12 and 14 proteoforms, respectively.
“Specifically, DDM allowed identification of an average of 96 proteoforms, in comparison to an average of 12 and 14 proteoforms using ProteaseMax and Invitrosol, respectively.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe analysis also identified brain acid soluble protein with N-myristoylation and multi-phosphorylated 60S acidic ribosomal protein P2.
“Following the same procedure, we also identified brain acid soluble protein with N-myristoylation (Figure S7), and multi-phosphorylated 60S acidic ribosomal protein P2 (Figure S8).”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationTop-down proteomics avoids proteolytic digestion and directly characterizes intact proteins, enabling complete characterization of proteoforms.
“In contrast, top-down proteomics bypasses the proteolytic step and characterizes intact proteins directly, allowing for complete characterization of “proteoforms”, which represent the collection of all possible modified forms of single genes.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [1][2]2][3]
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PremisecitationA major technical gap in top-down proteomics is that it cannot analyze low amounts of biological material, which restricts its use for rare cells, fine needle aspiration biopsies, and tissue substructures.
“However, many challenges remain especially when dealing with scant biological samples, such as isolated rare cells, fine needle aspiration biopsies, and tissue substructures.5,17”
Quote in the paperSubstantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationCombining nanoPOTS with FACS and LCM has enabled single-cell proteomics and spatially resolved proteomics tissue mapping.
“By combining nanoPOTS with fluorescence-activated cell sorting (FACS) [26][27][28] and laser capture microdissection (LCM), 29,30 single cell proteomics and spatially resolved proteomics tissue mapping have been demonstrated.”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationLabile MS-compatible detergents can be degraded before MS analysis to reduce interference.
“Another class of MScompatible detergents are labile molecules that can be degraded before MS to minimize interference.[22][23][24]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [22][23][24]
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PremisecitationProton transfer reaction methods recently introduced in top-down proteomics lower charge states and simplify mass spectra, which improves analysis of complex intact protein mixtures.
“Recent implementations of proton transfer reaction in top-down proteomics have allowed to reduce the charge states and simplify mass spectra, leading to significant improvements in analysis of complex intact protein mixtures.12,44,45”
Quote in the paperNot a substantial claim0 of 3 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationA prior CZE-MS study identified about 800 proteoforms from only 250 ng of proteins.
“A recent study using CZE-MS indicated ~800 proteoforms could be identified from only 250 ng of proteins.19”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [19] source
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PremisecitationPEG 20000 is expected to reduce adsorptive loss of proteins through competitive binding.
“PEG 20000 is expected to reduce adsorptive loss of proteins via competitive binding.40”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [40] source
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PremisecitationDDM is a nonionic detergent that has been shown to be effective for nanoPOTS-based bottom-up proteomics.
“DDM is a nonionic detergent, which has been demonstrated as an effective detergent for nanoPOTS-based bottom-up proteomics.[26][27][28]30”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [26][27][28]30
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PremisecitationProteaseMax, DDM, and Invitrosol have all been shown to improve recovery and coverage in bottom-up proteomics.
“All three detergents have shown improved recovery and coverage for bottom-up proteomics.36,37”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationNonionic detergents such as DDM are generally considered MS-compatible and have been used to maintain higher-order structures of membrane protein complexes in native MS.
“Nonionic detergents such as n-Dodecyl-β-D-Maltoside (DDM) have been generally considered MS-compatible, and have even been used for maintaining higher-order structures of membrane protein complexes in native MS.21”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [21] source
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Interpretationown dataThe nanoPOTS-based sample preparation successfully extracted and recovered large proteoforms, but the current Orbitrap MS did not identify them.
“This suggested that large proteoforms were successfully extracted and recovered in the nanoPOTS-based sample preparation process but not identified using the current Orbitrap MS.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAdvances in sample preparation, separation, instrumentation, and software have made it possible to identify and quantify proteoforms from various biological samples in depth.
“Ongoing technology developments in sample preparation, separation, instrumentation, and software have enabled the indepth identification and precise quantitation of proteoforms from various biological samples.[5][6][7][8][9][10][11][12][13][14]”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [5][6][7][8][9][10][11][12][13][14]
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PremisecitationThe nanoPOTS platform enabled identification and quantitation of about 3000 proteins from 10 HeLa cells and about 2400 proteins from a single human pancreatic islet thin section.
“The nanoPOTS platform enabled the identification and quantitation of ~3000 proteins from 10 HeLa cells and ~2400 proteins from a single human pancreatic islet thin section.31”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [31] source
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Classified as probably not a substantial claim. May combine two claims.
Study design and conductnone givenThe enclosed chamber prevented droplet evaporation and efficiently minimized protein contamination from the laboratory environment.
“The enclosed chamber not only prevented droplet evaporation, but also efficiently minimized protein contamination from the lab environment.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationUsing nonionic detergent as a universal carrier to improve protein recovery has been well demonstrated previously.
“Indeed, the use of non-ionic detergent as a universal carrier to improve protein recovery has been well demonstrated previously.35,38,39”
Quote in the paperNot a substantial claim0 of 3 sources checkedMay combine two claims.
Interpretationown dataThe combination of urea and DDM appears to facilitate both protein extraction and sample recovery for samples containing small amounts of intact proteins.
“Based on the increase in total number of proteins and proteoforms detected, the combination of urea and DDM appears to efficiently facilitate both protein extraction and sample recovery for samples containing small amount of intact proteins.”
Quote in the paperSubstantial claimMay combine two claims.
PremisecitationSDS solubilizes many proteins including membrane proteins, but it suppresses ions and is generally incompatible with downstream MS, and removing it can cause substantial protein loss.
“For instance, a widely used detergent sodium dodecyl sulfonate (SDS) is extremely efficient in solubilizing a variety of proteins including membrane proteins most notably for gel electrophoresis.20 However, SDS results in ion suppression and is generally con-sidered incompatible with downstream MS.The extra steps required to remove SDS can also cause significant loss of protein.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [20] source
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PremisecitationDifferent proteoforms from the same gene can have distinct functions and activities because they respond differently in gene regulation and cell signaling.
“Different proteoforms from the same gene can have distinct functions and activities, because of their different responses in gene regulation and cell signaling.4”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [4] source
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InterpretationreasoningThe higher number of identifications with DDM is probably not caused by greater protein extraction efficiency; instead, DDM's advantage is attributed to reducing protein adsorption on well surfaces more effectively than the other buffers.
“Therefore, the increase in the number of identifications is not likely due to an increased protein extraction efficiency.Rather, we ascribe the superior performance of DDM to a more efficient reduction of protein adsorption on well surfaces than other extraction buffers explored.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationCalmodulin, UniProt accession P0DP23, is known to undergo acetylation at K95 or trimethylation at K116.
“This protein (UniProt accession: P0DP23) is known to be acetylated at K95, or trimethylated at K116. 49”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [49] source
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Study design and conductcitationThe custom filtering step reduced redundancy in proteoform counts that is typically caused by deisotoping errors, but it also merged proteoforms differing only slightly in mass, such as disulfide or deamidation variants, and positional isoforms likely to co-elute within 15 minutes.
“This filtering step of the TopPIC result reduced the redundancy in proteoform counts, which is typically related to deisotoping errors (i.e., masses can differ by a few Da due to mis-assigned monoisotopic peaks).5 However, this also resulted in merging the proteoforms that differ in mass only slightly (e.g., disulfide, deamidation), as well as positional isoforms, which are likely to co-elute with”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [5] source
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PremisecitationReversed phase liquid chromatography with short alkyl C2 and C4 bonded phases provides both high-resolution separation and high sample recovery.
“One of the efficient separation techniques is reversed phase liquid chromatography (RPLC) using short alkyl (C2 and C4) bonded phases, which has been demonstrated to provide both high resolution separation and high sample recovery.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [18] source
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PremisecitationnanoPOTS was previously introduced as an effective approach for bottom-up proteomic analysis of limited numbers of cells, down to a single mammalian cell.
“The nanodroplet array, referred to nanoPOTS (Nanodroplet Processing in One pot for Trace Samples), was previously introduced as an effective approach for bottom-up proteomic analysis of limited number of cells and down to a single mammalian cell.[26][27][28][29][30][31]”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26][27][28][29][30][31]
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PremisereasoningSample recovery can be substantially improved by preparing samples in nanodroplets smaller than 200 nL, probably because surface adsorption of proteins and peptides in nanowells is minimized.
“We demonstrated that sample recovery can be significantly improved by preparation inside nanodroplets with volumes smaller than 200 nL, likely due to minimization of surface adsorption of proteins and peptides in nanowells.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [26][27][28][29][30][31]
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InterpretationreasoningThe marginal improvement from PEG 20000 for 10-20 kDa proteoforms may be due to reduced nonspecific binding, which improves recovery of larger proteoforms.
“On the other hand, PEG 20000 showed a marginal improvement for identification of proteoforms with mass 10-20 kDa possibly due to the reduced nonspecific binding (i.e.improved recovery of larger proteoforms).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S2 (checkable).
InterpretationreasoningImproving both proteome coverage depth and quantitation accuracy will require additional technology development and refinement.
“further technology developments and refinements are clearly required to improve both, the depth of proteome coverage and the accuracy of quantitation.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningThe gains from DDM and urea are ascribed to less non-specific surface adsorption and better protein recovery for DDM, and to disruption of noncovalent complexes and release of intact proteins for urea.
“These enhancements were attributed to efficient reduction of non-specific adsorption on surfaces and improved protein recovery (DDM), together with efficient disruption of noncovalent complexes (urea) and release of intact proteins for downstream LC-MS analysis.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationHuman cells are estimated to contain more than one million proteoforms.
“Given that over a million proteoforms are estimated to be present in human cells, 57”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites [57] source
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The paper relies on this without support.
The number of identified proteoforms or proteins is a valid proxy for protein extraction and recovery efficiency.
“Improvements were judged by whether the number of protein or proteoform identifications increased upon addition of these reagents.”
The paper relies on this without support.
HeLa cells are representative of mammalian cells for the nanoPOTS top-down proteomics platform.
“In this study, we demonstrated ability to perform top-down proteomic analysis of small number of mammalian cells by scaling down the sample processing using the nanoPOTS platform.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
nanoPOTS is promising for highly sensitive top-down proteomics potentially including single-cell analysis, but most protein identifications were limited to sizes below 10 kDa.
“The nanoPOTs technology, although still highly specialized, shows enormous promise to enable highly sensitive top-down proteomics (potentially even single-cell top-down analysis), but the majority of protein identifications were limited to sizes <10 kDa.”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [38]
DDM combined with urea enabled extraction and identification of ~170-620 proteoforms from ~70-770 HeLa cells.
“They found that a combination of dodecyl-β-D-maltopyranoside (DDM) with urea enabled the extraction and identification of ~170–620 proteoforms from as little as ~70–770 HeLa cells [38].”
From Top-down proteomics: challenges, innovations, and applications in basic and clinical research (2020) · cites this paper as [38]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
The listed buffers omit a claimed PEG test condition
MinorThe paper says PEG 20000 was added to both 0.5% and 0.1% DDM, but its list of the seven buffers includes PEG only with 0.1% DDM. No 0.5% DDM plus PEG condition is listed.
“First, we tested the addition of 0.01% PEG 20000 into 0.5% DDM and 0.1% DDM in the extraction buffer.”
“5 0.5% DDM 6 0.1% DDM + 0.01% PEG 20000 7 0.1% DDM + 8 M urea”
Cites
A cross-platform toolkit for mass spectrometry and proteomics
Chambers, M., MacLean, B., Burke, R., Amodei, D., Ruderman, D., Neumann, S., Gatto, L., Fischer, B., Pratt, B. S., Egertson, J. D., et al. (2012) · Nature Biotechnology · doi:10.1038/nbt.2377 · Cited by 4,042 · see citing works
Protein Analysis by Shotgun/Bottom-up Proteomics
Zhang, Y., Fonslow, B. R., Shan, B., Baek, M.-C., & Yates, J. R. (2013) · Chemical Reviews · doi:10.1021/cr3003533 · Cited by 1,491 · see citing works
Marty, M. T., Baldwin, A. J., Marklund, E. G., Hochberg, G. K. A., Benesch, J. L. P., & Robinson, C. V. (2015) · Analytical Chemistry · doi:10.1021/acs.analchem.5b00140 · Cited by 1,000 · see citing works
How many human proteoforms are there?
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Activated Ion-Electron Transfer Dissociation Enables Comprehensive Top-Down Protein Fragmentation
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Capillary HILIC-MS: A New Tool for Sensitive Top-Down Proteomics
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Self-citationTop-Down LESA Mass Spectrometry Protein Analysis of Gram-Positive and Gram-Negative Bacteria
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The Human Proteoform Project: Defining the human proteome
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Self-citationApplications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research
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High sensitivity top–down proteomics captures single muscle cell heterogeneity in large proteoforms
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Self-citationRecent advances in top‐down proteome sample processing ahead of MS analysis
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Proteoform‐Selective Imaging of Tissues Using Mass Spectrometry**
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Improved Identification of Proteoforms in Top-Down Proteomics Using FAIMS with Internal CV Stepping.
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Microproteomic sample preparation
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Hanging drop sample preparation improves sensitivity of spatial proteomics
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Self-citationAutomated imaging and identification of proteoforms directly from ovarian cancer tissue
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Single-cell omic molecular profiling using capillary electrophoresis-mass spectrometry
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Trends in Mass Spectrometry-Based Single-Cell Proteomics
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Proteomics-Based Identification of Dysregulated Proteins in Breast Cancer
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Biological mass spectrometry enables spatiotemporal ‘omics: From tissues to cells to organelles
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Capillary Electrophoresis–Mass Spectrometry for Top-Down Proteomics
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Self-citationSingle‐cell proteomics by mass spectrometry: Advances and implications in cancer research
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Measuring expression heterogeneity of single-cell cytoskeletal protein complexes
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Measuring expression heterogeneity of single-cell cytoskeletal protein complexes
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Spatial top-down proteomics for the functional characterization of human kidney
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Addressing Cellular Heterogeneity in Cancer through Precision Proteomics
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The Human Proteoform Project: A Plan to Define the Human ProteomePreprint
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Self-citationAdvances in proteomic phenotyping of microglia in neurodegeneration
Sunna, S. N., Bowen, C. A., Ramelow, C. C., Santiago, J. V., Kumar, P., & Rangaraju, S. (2023) · PROTEOMICS · doi:10.1002/pmic.202200183 · Cited by 12 · see citing works
TopPICR: A Companion R Package for Top-Down Proteomics Data Analysis
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The Proteoform Landscape of Current Top-Down ProteomicsPreprint
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- Authors
- Zhou, M., Uwugiaren, N., Williams, S. M., Moore, R. J., Zhao, R., Goodlett, D., Dapic, I., Paša-Tolić, L., & Zhu, Y.
- Affiliations
- Pacific Northwest National Laboratory (US)
- University of Gdańsk (PL)
- University of Maryland, Baltimore (US)
- International Centre for Cancer Vaccine Science, University of Gdansk, Gdansk, Poland
- Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, United States
- Corresponding author
- Zhu, Ying
- Venue
- Analytical Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2020-05-06
- Volume
- 92
- Issue
- 10
- Pages
- 7087-7095
- ISSN
- 0003-2700 (ISSN-L)
- 1520-6882
- OpenAlex
- W3023709981
- Cited by
- 62
- References
- 57
- Citations by year
- 2026: 7 · 2025: 10 · 2024: 9 · 2023: 12 · 2022: 9 · 2021: 11 · 2020: 4
- FWCI
- 3.43
- Type
- journal article
- Language
- en
- Open access
- green
- www.osti.gov
- Licence
- Locations
- Analytical Chemistry (publishedVersion)
- PubMed (publishedVersion)
- OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) (submittedVersion)
- Funders
- Biological and Environmental Research (grid.436923.9)
- European Regional Development Fund (International Research Agendas program of the Foun)
- Topics
- Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
- Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Advanced Biosensing Techniques and Applications
- Keywords
- top-down proteomics, microfluidic sample preparation, urea denaturation, protein extraction, proteoforms, post-translational modification, acetylation, myristoylation, iron binding, HeLa cells
- MeSH terms
- HeLa Cells, Humans, Maltose, Neoplasm Proteins, Particle Size, Surface Properties, Tumor Cells, Cultured, Urea, Proteomics, Microfluidic Analytical Techniques, Nanoparticles
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