Laser‐induced liquid bead ion desorption‐MS of protein complexes from blue‐native gels,a sensitive top‐down proteomic approach

We have developed an experimental approach that combines two powerful methods for proteomic analysis of large membrane protein complexes: blue native electrophoresis (BNE or BN‐PAGE) and laser‐induced liquid bead ion desorption (LILBID) MS. Protein complexes were separated by BNE and eluted from the gel. The masses of the constituents of the multiprotein complexes were obtained by LILBID MS, a detergent‐tolerant method that is especially suitable for the characterisation of membrane proteins. High sensitivity and small sample volumes required for LILBID MS resulted in low demands on sample quantity. Eluate from a single band allowed assessing the mass of an entire multiprotein complex and its subunits. The method was validated with mitochondrial NADH:ubiquinone reductase from Yarrowia lipolytica. For this complex of 947 kDa, typically 30 μg or 32 pmol were sufficient to obtain spectra from which the subunit composition could be analysed. The resolution of this electrophoretic small‐scale approach to the purification of native complexes was improved markedly by further separation on a second dimension of BNE. Starting from a subcellular fraction obtained by differential centrifugation, this allowed the purification and analysis of the constituents of a large multiprotein complex in a single LILBID spectrum.     

Unraveling tobacco BY-2 protein complexes with BN PAGE/LC-MS/MS and clustering methods

To understand physiological processes, insight into protein complexes is very important. Through a combination of blue native gel electrophoresis and LC-MS/MS, we were able to isolate protein complexes and identify their potential subunits from Nicotiana tabacum cv. Bright Yellow-2. For this purpose, a bioanalytical approach was used that works without a priori knowledge of the interacting proteins. Different clustering methods (e.g., k-means and hierarchical clustering) and a biclustering approach were evaluated according to their ability to group proteins by their migration profile and to correlate the proteins to a specific complex. The biclustering approach was identified as a very powerful tool for the exploration of protein complexes of whole cell lysates since it allows for the promiscuous nature of proteins. Furthermore, it searches for associations between proteins that co-occur frequently throughout the BN gel, which increases the confidence of the putative associations between co-migrating proteins. The statistical significance and biological relevance of the profile clusters were verified using functional gene ontology annotation. The proof of concept for identifying protein complexes by our BN PAGE/LC-MS/MS approach is provided through the analysis of known protein complexes. Both well characterized long-lived protein complexes as well as potential temporary sequential multi-enzyme complexes were characterized.     

Proteomics of mouse liver microsomes: Performance of different protein separation workflows for LC‐MS/MS

The mouse liver microsome proteome was investigated using ion trap MS combined with three separation workflows including SDS‐PAGE followed by reverse‐phase LC of in‐gel protein digestions (519 proteins identified); 2‐D LC of protein digestion (1410 proteins); whole protein separation on mRP heat‐stable column followed by 2‐D LC of protein digestions from each fraction (3‐D LC; 3703 proteins). The higher number of proteins identified in the workflow corresponded to the lesser percentage of run‐to‐run reproducibility. Gel‐based method yielded a number of predicted membrane proteins similar to LC‐based workflows.     

Micropreparative fractionation of the complexome by blue native continuous elution electrophoresis

The large‐scale analysis of protein complexes is an emerging challenge in the field of proteomics. Currently, there are few methods available for the fractionation of protein complexes that are compatible with downstream proteomic techniques. Here, we describe the technique of blue native continuous elution electrophoresis (BN‐CEE). It combines the features of blue native PAGE (BN‐PAGE) and continuous elution electrophoresis (CEE), generating liquid‐phase fractions of protein complexes of up to 800 kDa. The resulting complexes can be further analysed by BN‐PAGE, by SDS‐PAGE and/or by MS. This can help define the constituent proteins of many complexes and their stoichiometry. As BN‐CEE is also micropreparative, with a capacity to separate milligram quantities of protein complexes, it will assist the study of proteins of lower abundance. In this regard, the acrylamide concentration and elution rate during separation can be controlled to help ‘zoom in’ on particular high mass regions and thus complexes of interest. We illustrate the utility of the technique in the analysis of Saccharomyces cerevisiae cellular lysate.     

About three‐fourths of mouse proteins unexpectedly appear at a low position of SDS‐PAGE,often as additional isoforms,questioning whether all protein isoforms have been eliminated in gene‐knockout cells or organisms

Most genes in evolutionarily complex genomes are expressed to multiple protein isoforms, but there is not yet any simple high‐throughput approach to identify these isoforms. Using an oversimplified top‐down LC–MS/MS strategy, we detected, around the 26‐kD position of SDS‐PAGE, proteins produced from 782 genes in a Cdk4?/? mouse embryonic fibroblast cell line. Interestingly, only 213 (27.24%, about one‐fourth) of these 782 genes have their proteins with a theoretical molecular mass (TMM) 10% smaller or larger than 26 kD, that is, between 23 and 29 kD, the range set as allowed variation in SDS‐PAGE. These 213 proteins are considered as the wild type (WT). The remaining three‐fourths includes proteins from 66 (9.44%) genes with a TMM smaller than 23 kD and proteins from 503 (64.32%, nearly two‐thirds) genes with a TMM larger than 29 kD; these proteins are categorized into a larger‐group or a smaller‐group, respectively, for their appearance at a higher or lower position of SDS‐PAGE. For instance, at this 26‐kD position we detected proteins from the Rps27a, Snrpf, Hist1h4a, and Rps25 genes whose proteins' TMM is 8.6, 9.7, 11.4, and 13.7 kD, respectively, and detected proteins from the Plelc1 and Prkdc genes, whose largest isoform is 533.9 and 471.1 kD, respectively. We extrapolate that many of those proteins migrating unexpectedly in SDS‐PAGE may be isoforms besides the WT protein. Moreover, we also detected a Cdk4 protein in this Cdk4?/? cell line, thus wondering whether some of other gene‐knockout cells or organisms show similar incompleteness of the knockout.     

Analysis of membrane–protein complexes of the marine sulfate reducer Desulfobacula toluolica Tol2 by 1D blue native‐PAGE complexome profiling and 2D blue native‐/SDS‐PAGE

Sulfate‐reducing bacteria (SRB) obtain energy from cytoplasmic reduction of sulfate to sulfide involving APS‐reductase (AprAB) and dissimilatory sulfite reductase (DsrAB). These enzymes are predicted to obtain electrons from membrane redox complexes, i.e. the quinone‐interacting membrane‐bound oxidoreductase (QmoABC) and DsrMKJOP complexes. In addition to these conserved complexes, the genomes of SRB encode a large number of other (predicted) membrane redox complexes, the function and actual formation of which is unknown. This study reports the establishment of 1D Blue Native‐PAGE complexome profiling and 2D BN‐/SDS‐PAGE for analysis of the membrane protein complexome of the marine sulfate reducer Desulfobacula toluolica Tol2. Analysis of normalized score profiles of >800 proteins in combination with hierarchical clustering and identification of 2D BN‐/SDS‐PAGE separated spots demonstrated separation of membrane complexes in their native form, e.g. ATP synthase. In addition to the QmoABC and DsrMKJOP complexes, other complexes were detected that constitute the basic membrane complexome of D. toluolica Tol2, e.g. transport proteins (e.g. sodium/sulfate symporters) or redox complexes involved in Na⁺‐based bioenergetics (RnfABCDEG). Notably, size estimation indicates dimer and quadruple formation of the DsrMKJOP complex in vivo. Furthermore, cluster analysis suggests interaction of this complex with a rhodanese‐like protein (Tol2_C05230) possibly representing a periplasmic electron transfer partner for DsrMKJOP.     

Helicobacter pylori proteomics by 2‐DE/MS, 1‐DE‐LC/MS and functional data mining

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea‐solubilized proteins by 2‐DE/MS (data set 2‐DE) and by 1‐DE‐LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1‐DE‐LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research ( http://www.mpiib‐berlin.mpg.de/2D‐PAGE/ ), which gives access to raw mass spectra (MALDI‐TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT ( http://webclu.bio.wzw.tum.de/prompt/ ) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2‐DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2‐DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea‐insoluble proteins and makes them accessible for separation by SDS‐PAGE and LC. The 2‐DE/MS analysis with urea‐solubilized proteins and the 1‐DE‐LC/MS analysis with the urea‐insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT‐generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.     

Analysis of outer membrane porin complexes of Neisseria meningitidis in wild‐type and specific knock‐out mutant strains

The structure of the porin complexes of Neisseria meningitidis was assessed in the vaccine strain H44/76 and its homologous mutants lacking the main porins (PorA and PorB) and other outer membrane (OM) components (RmpM and FetA). The analysis using 1‐D blue native (BN) electrophoresis, 2‐D BN/SDS‐PAGE and 2‐D diagonal electrophoresis, followed by LC/MS‐MS (for 1‐D gels) or MALDI‐TOF (for 2‐D gels) revealed at least six porin complexes in the wild‐type strain with molecular masses (MW) ranging from 145 to 195 kDa and variable composition: The two higher MW complexes are formed by PorA, PorB and RmpM, the following three are formed by PorA and PorB, and the lower MW one is formed by only PorB. Complexes in the mutants lacking either PorA, PorB or RmpM, but not those in the mutant lacking FetA, were alterered respect to those in the wild‐type strain. The most evident alteration was seen in the mutant lacking PorB, in which PorA formed only a high MW complex (≈?800 kDa). Our results suggest that PorA and PorB could form a ‘basic’ template for the transportation systems in the OM of the meningococci. Other proteins (such as RmpM) could be transiently associated to the porin complexes, depending on the specific tranport needs at different stages of the meningococcal life cycle, resulting in a dynamic net of pores of variable composition.     

Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling

Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome‐wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label‐free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co‐IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co‐migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co‐elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC–MS analysis for system‐wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.     

Proteomic analysis on the temperature‐dependent complexes in Thermoanaerobacter tengcongensis

It is generally accepted that protein complexes play an active role in avoiding the protein degradation of the thermophiles. Thermoanaerobacter tengcongensis was cultured at three different temperatures (55, 75 and 80°C) and the extracts of protein complexes were prepared. Through blue native PAGE, the changes of the relative band volumes in response to different temperatures were semi‐quantitatively compared and six temperature‐dependent bands were obtained. These bands were excised, digested with trypsin and then analyzed with MS for the identification of protein components. With the combination of the proteins identified by LC MS/MS and MALDI TOF/TOF MS, a total of 92 unique proteins were ascertained in these complexes. Besides, some protein components were examined with Western blot, which gave us insights into the survival mechanism of thermophiles. These included (i) the composition of complex at 80°C was significantly different from that at the other two temperatures; (ii) HSPs presented in all temperature‐dependent complexes; (iii) several proteins associated with the functional pathways existed in the same complexes, indicating that the complex structure provided facility for the functional efficiency.     

iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

Low-SDS Blue native PAGE

SDS normally is strictly avoided during Blue native (BN) PAGE because it leads to disassembly of protein complexes and unfolding of proteins. Here, we report a modified BN-PAGE procedure, which is based on low-SDS treatment of biological samples prior to native gel electrophoresis. Using mitochondrial OXPHOS complexes from Arabidopsis as a model system, low SDS concentrations are shown to partially dissect protein complexes in a very defined and reproducible way. If combined with 2-D BN/SDS-PAGE, generated subcomplexes and their subunits can be systematically investigated, allowing insights into the internal architecture of protein complexes. Furthermore, a 3-D BN/low-SDS BN/SDS-PAGE system is introduced to facilitate structural analysis of individual protein complexes without their previous purification.     

Phosphoprotein profiling by PA-GeLC-MS/MS

A significant consequence of protein phosphorylation is to alter protein-protein interactions, leading to dynamic regulation of the components of protein complexes that direct many core biological processes. Recent proteomic studies have populated databases with extensive compilations of cellular phosphoproteins and phosphorylation sites and a similarly deep coverage of the subunit compositions and interactions in multiprotein complexes. However, considerably less data are available on the dynamics of phosphorylation, composition of multiprotein complexes or that define their interdependence. We describe a method to identify candidate phosphoprotein complexes by combining phosphoprotein affinity chromatography, separation by size, denaturing gel electrophoresis, protein identification by tandem mass spectrometry, and informatics analysis. Toward developing phosphoproteome profiling, we have isolated native phosphoproteins using a phosphoprotein affinity matrix, Pro-Q Diamond resin (Molecular Probes-Invitrogen). This resin quantitatively retains phosphoproteins and associated proteins from cell extracts. Pro-Q Diamond purification of a yeast whole cell extract followed by 1-D PAGE separation, proteolysis and ESI LC-MS/MS, a method we term PA-GeLC-MS/MS, yielded 108 proteins, a majority of which were known phosphoproteins. To identify proteins that were purified as parts of phosphoprotein complexes, the Pro-Q eluate was separated into two fractions by size, <100 kDa and >100 kDa, before analysis by PAGE and ESI LC-MS/MS and the component proteins queried against databases to identify protein-protein interactions. The <100 kDa fraction was enriched in phosphoproteins indicating the presence of monomeric phosphoproteins. The >100 kDa fraction contained 171 proteins of 20-80 kDa, nearly all of which participate in known protein-protein interactions. Of these 171, few are known phosphoproteins, consistent with their purification by participation in protein complexes. By comparing the results of our phosphoprotein profiling with the informational databases on phosphoproteomics, protein-protein interactions and protein complexes, we have developed an approach to examining the correlation between protein interactions and protein phosphorylation.     

Improving basic and membrane protein MS detection of the culture filtrate proteins from Mycobacterium tuberculosis H37Rv by biomimetic affinity prefractionation

The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines, and diagnostics. The constituents of the M. tuberculosis CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel‐based prefractionation is usually performed before MS analysis. In this study, we describe a novel prefractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six‐column cascade. The LC‐MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first‐time identifications. Notably, ～1/3 (20/61) of these novel CFPs are membrane proteins, among which nine proteins have 2–14 transmembrane domains. In addition, ～1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that biomimetic affinity chromatography prefractionation markedly improves protein detection by LC‐MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased.     

Cross‐species comparison of mammalian saliva using an LC–MALDI based proteomic approach

Despite the importance of saliva in the regulation of oral cavity homeostasis, few studies have been conducted to quantitatively compare the saliva of different mammal species. Aiming to define a proteome signature of mammals’ saliva, an in‐depth SDS‐PAGE–LC coupled to MS/MS (GeLC–MS/MS) approach was used to characterize the saliva from primates (human), carnivores (dog), glires (rat and rabbit), and ungulates (sheep, cattle, horse). Despite the high variability in the number of distinct proteins identified per species, most protein families were shared by the mammals studied with the exception of cattle and horse. Alpha‐amylase is an example that seems to reflect the natural selection related to digestion efficacy and food recognition. Casein protein family was identified in all species but human, suggesting an alternative to statherin in the protection of hard tissues. Overall, data suggest that different proteins might assure a similar role in the regulation of oral cavity homeostasis, potentially explaining the specific mammals’ salivary proteome signature. Moreover, some protein families were identified for the first time in the saliva of some species, the presence of proline‐rich proteins in rabbit's saliva being a good example.     

Secreted phosphoprotein‐24 kDa (Spp24) attenuates BMP‐2‐stimulated Smad 1/5 phosphorylation and alkaline phosphatase induction and was purified in a protective complex with alpha2‐Macroglobulins From Serum

Secreted phosphoprotein‐24 kDa (Spp24) binds cytokines of the bone morphogenetic protein/transforming growth factor‐β (BMP/TGFβ) superfamily and is one of the most abundant serum phosphoproteins synthesized by the liver. Little is known about how Spp24 binding affects BMP signal transduction and osteoblastic differentiation or how this labile protein is transported from the liver to remote tissues, such as bone. When Spp24 was administered to W‐20‐17 mesenchymal stem cells with rhBMP‐2, short‐term Smad1/5 phosphorylation was inhibited, intermediate‐term alkaline phosphatase (ALP) induction was blunted, and long‐term mineralization was unaffected. This supports the hypothesis that Spp24 proteolysis restricts the duration of its regulatory effects, but offers no insight into how Spp24 is transported intact from the liver to bone. When Spp24 was immunopurified from serum and subjected to native PAGE and Western blotting, a high molecular weight band of >500 kDa was found. Under reducing SDS–PAGE, a 24 kDa band corresponding to monomeric Spp24 was liberated, suggesting that Spp24 is bound to a complex linked by disulfide bonds. However, such a complex cannot be disrupted by 60 mM EDTA under non‐reducing condition or in purification buffers containing 600 mM NaCl and 0.1% Tween‐20 at pH 2.7–8.5. LC–MS/MS analysis of affinity‐purified, non‐reducing SDS–PAGE separated, and trypsin digested bands showed that the Spp24 was present in a complex with three α₂‐macroglobulins (α₂‐macroglobulin [α₂M], pregnancy zone protein [PZP] and complement C3 [C3]), as well as ceruloplasmin and the protease inhibitor anti‐thrombin III (Serpin C1), which may protect Spp24 from proteolysis. J. Cell. Biochem. 114: 378–387, 2013. © 2012 Wiley Periodicals, Inc.     

Isoforms of wild type proteins often appear as low molecular weight bands on SDS‐PAGE

Immunoblotting, after polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS‐PAGE), is a technique commonly used to detect specific proteins. SDS‐PAGE often results in the visualization of protein band(s) in addition to the one expected based on the theoretical molecular mass (TMM) of the protein of interest. To determine the likelihood of additional band(s) being nonspecific, we used liquid chromatography – mass spectrometry to identify proteins that were extracted from bands with the apparent molecular mass (MM) of 40 and 26 kD, originating from protein extracts derived from non‐malignant HEK293 and cancerous MDA‐MB231 (MB231) cells separated using SDS‐PAGE. In total, approximately 57% and 21% of the MS/MS spectra were annotated as peptides in the two cell samples, respectively. Moreover, approximately 24% and 36.2% of the identified proteins from HEK293 and MB231 cells matched their TMMs. Of the identified proteins, 8% from HEK293 and 26% from MB231 had apparent MMs that were larger than predicted, and 67% from HEK293 and 37% from MB231 exhibited smaller MM values than predicted. These revelations suggest that interpretation of the positive bands of immunoblots should be conducted with caution. This study also shows that protein identification performed by mass spectrometry on bands excised from SDS‐PAGE gels could make valuable contributions to the identification of cancer biomarkers, and to cancer‐therapy studies.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Intact protein profiling in breast cancer biomarker discovery: Protein identification issue and the solutions based on 3D protein separation,bottom‐up and top‐down mass spectrometry

Proteomics profiling of intact proteins based on MALDI‐TOF MS and derived platforms has been used in cancer biomarker discovery studies. This approach suffers from a number of limitations such as low resolution, low sensitivity, and that no knowledge is available on the identity of the respective proteins in the discovery mode. Nevertheless, it remains the most high‐throughput, untargeted mode of clinical proteomics studies to date. Here we compare key protein separation and MS techniques available for protein biomarker identification in this type of studies and define reasons of uncertainty in protein peak identity. As a result of critical data analysis, we consider 3D protein separation and identification workflows as optimal procedures. Subsequently, we present a new protocol based on 3D LC‐MS/MS with top‐down at high resolution that enabled the identification of HNRNP A2/B1 intact peptide as correlating with the estrogen receptor expression in breast cancer tissues. Additional development of this general concept toward next generation, top‐down based protein profiling at high resolution is discussed.