基于亲和色谱的肺癌细胞磷酸化蛋白质组研究及其应用

磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti⁴⁺)螯合IMAC材料(Ti⁴⁺-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti⁴⁺-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti⁴⁺-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti⁴⁺-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。     

An ultra-fast and highly efficient multiple proteases digestion strategy using graphene-oxide-based immobilized protease reagents

Highly efficient and rapid proteolytic digestion of proteins into peptides is a crucial step in shotgun-based proteome-analysis strategy.Tandem digestion by two or more proteases is demonstrated to be helpful for increasing digestion efficiency and decreasing missed cleavages,which results in more peptides that are compatible with mass-spectrometry analysis.Compared to conventional solution digestion,immobilized protease digestion has the obvious advantages of short digestion time,no self-proteolysis,and reusability.We proposed a multiple-immobilized proteases-digestion strategy that combines the advantages of the two digestion strategies mentioned above.Graphene-oxide(GO)-based immobilized trypsin and endoproteinase Glu-C were prepared by covalently attaching them onto the GO surface.The prepared GO-trypsin and GO-Glu-C were successfully applied in standard protein digestion and multiple immobilized proteases digestion of total proteins of Thermoanaerobacter tengcongensis.Compared to 12-hour solution digestion using trypsin or Glu-C,14%and 7%improvement were obtained,respectively,in the sequence coverage of BSA by one-minute digestion using GO-trypsin and GO-Glu-C.Multiple immobilized-proteases digestion of the total proteins of Thermoanaerobacter tengcongensis showed 24.3%and 48.7%enhancement in the numbers of identified proteins than was obtained using GO-trypsin or GO-Glu-C alone.The ultra-fast and highly efficient digestion can be contributed to the high loading capacity of protease on GO,which leads to fewer missed cleavages and more complete digestion.As a result,improved protein identification and sequence coverage can be expected.     

Development of phosphopeptide enrichment techniques for phosphoproteome analysis

Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications. The analysis of protein phosphorylation is very challenging due to its highly dynamic nature and low stoichiometry. In this article, recent techniques developed for phosphoproteome analysis are reviewed with an emphasis on the new developments in this field in China. To improve the performance of phosphoproteome analysis, many novel methods, either by application of new separation mechanisms or by adoption of new separation materials, were developed to specifically enrich phosphopeptides from complex protein digests. A series of new materials, including nanostructure materials, magnetic materials, and monolithic materials, were applied to prepare immobilized affinity chromatography or metal oxide affinity chromatography to improve the performance of phosphopeptide enrichment. Besides, new software tools were also developed to validate phosphopeptide identification and predict kinase specific phosphorylation sites.     

Assessment of microwave-assisted enzymatic digestion by measuring glycated hemoglobin A1c by mass spectrometry

Enzymatic digestion of proteins and analysis of the resulting peptides by mass spectrometry is an established approach in proteomics and in clinical and environmental chemistry. The long digestion times of several hours prevent the fast turnover of samples and results. Qualitative applications showed that microwave radiation profoundly shortens enzymatic digestion. However, its usefulness for quantitative applications had not been assessed. In this study, the microwave-assisted enzymatic digestion of hemoglobin at different temperatures, buffer concentrations, and digestion times was assessed and compared with conventional digestion for the proteolytic enzymes trypsin and Glu-C. A microwave-assisted enzymatic digestion method optimized for digestion time and temperature was applied for the analysis of glycated hemoglobin HbA1c and compared with a reference method. Using trypsin, complete digestion was obtained at 50 degrees C within 20 min. Under these conditions, the digestion efficiency was 20% higher than with conventional trypsin digestion. These effects were not observed with Glu-C as enzyme, probably because of the decreased stability of Glu-C at elevated temperatures in comparison with the trypsin used. The comparison of the optimized microwave-assisted digestion method using trypsin with the reference method for HbA1c using Glu-C gave a close correlation in the results (R2: 0.996). A significant bias of 0.33% HbA1c was observed, with higher values obtained with the microwave-assisted tryptic digest; this finding might have resulted from the use of a different enzyme. This study showed that microwave-assisted enzymatic digestion can substantially reduce digestion times to minutes and can be used in qualitative as well as quantitative applications.     

Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides

Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga³⁺ chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga³⁺-ATP-MNPs). The high specificity of Ga³⁺-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000. Ga³⁺-ATP-MNPs were also successfully applied for the phosphoproteome analysis of rat liver mitochondria, resulting in the identification of 193 phosphopeptides with 331 phosphorylation sites from 158 phosphoproteins. In other words, 54.4% of the phosphopeptides trapped by Ga³⁺-ATP-MNPs were observed with more than one phosphorylated sites, resulting in significant improvement on the identification of peptides with multi-phosphorylated sites. The high specificity of Ga³⁺-ATP-MNPs towards multi-phosphopeptides may be due to the synergistic effect of the strong hydrophilic surface functionalized by ATP and the proper chelating strength provided by Ga³⁺. Moreover, the unique magnetic core of Ga³⁺-ATP-MNPs also facilitates the isolation process and on-plate enrichment for direct MALDI MS analysis with limit of detection as low as 30 amol. This new affinity-based protocol is expected to provide a powerful approach for characterizing multiple phosphorylation sites on proteins in complex and dilute analytes, which may be explored as complementary technique for improving the coverage of phosphoproteome.     

Global quantification of phosphoproteins combining metabolic labeling and gel‐based proteomics in B. pumilus

Differential proteomics targeting the protein abundance is commonly used to follow changes in biological systems. Differences in localization and degree of post‐translational modifications of proteins including phosphorylations are of tremendous interest due to the anticipated role in molecular regulatory processes. Because of their particular low abundance in prokaryotes, identification and quantification of protein phosphorylation is traditionally performed by either comparison of spot intensities on two‐dimensional gels after differential phosphoprotein staining or gel‐free by stable isotope labeling, sequential phosphopeptide enrichment and following LC‐MS analysis. In the current work, we combined in a proof‐of‐principle experiment these techniques using ¹⁴N/¹⁵N metabolic labeling with succeeding protein separation on 2D gels. The visualization of phosphorylations on protein level by differential staining was followed by protein identification and determination of phosphorylation sites and quantification by LC‐MS/MS. This approach should avoid disadvantages of traditional workflows, in particular the limited capability of peptide‐based gel‐free methods to quantify isoforms of proteins. Comparing control and stress conditions allowed for relative quantification in protein phosphorylation in Bacillus pumilus exposed to hydrogen peroxide. Altogether, we quantified with this method 19 putatively phosphorylated proteins.     

Automated phosphoproteome analysis for cultured cancer cells by two-dimensional nanoLC-MS using a calcined titania/C18 biphasic column.

We have developed an on-line automated system for phosphoproteome analysis using titania-based phosphopeptide enrichment followed by nanoLC-MS/MS. Titania beads were prepared by calcination of commercial chromatographic titania beads at 800 degrees C to convert the crystalline structure. The obtained rutile-form titania exhibited higher selectivity in phosphopeptide enrichment than commercial titania, even in the absence of a competitive chelating reagent for non-phosphopeptides. For phosphoproteome analysis of human cervical cancer HeLa cells, tryptic digests of the cell extracts were directly injected into this on-line system, and 696 non-redundant phosphopeptides with 671 unambiguously determined phosphorylation sites, derived from 512 phosphoproteins, were successfully identified. This is the first successful application of an on-line automated phosphoproteome analysis system to complex biological samples.     

Rapid fabrication of glass/PDMS hybrid µIMER for high throughput membrane proteomics

Mass spectrometry (MS) based proteomics has brought a radical approach to systems biology, offering a platform to study complex biological functions. However, key proteomic technical challenges remain, mainly the inability to characterise the complete proteome of a cell due to the thousands of diverse, complex proteins expressed at an extremely wide concentration range. Currently, high throughput and efficient techniques to unambiguously identify and quantify proteins on a proteome-wide scale are in demand. Miniaturised analytical systems placed upstream of MS help us to attain these goals. One time-consuming step in traditional techniques is the in-solution digestion of proteins (4-20 h). This also has other drawbacks, including enzyme autoproteolysis, low efficiency, and manual operation. Furthermore, the identification of α-helical membrane proteins has remained a challenge due to their high hydrophobicity and lack of trypsin cleavage targets in transmembrane helices. We demonstrate a new rapidly produced glass/PDMS micro Immobilised Enzyme Reactor (μIMER) with enzymes covalently immobilised onto polyacrylic acid plasma-modified surfaces for the purpose of rapidly (as low as 30 s) generating peptides suitable for MS analysis. This μIMER also allows, for the first time, rapid digestion of insoluble proteins. Membrane protein identification through this method was achieved after just 4 min digestion time, up to 9-fold faster than either dual-stage in-solution digestion approaches or other commonly used bacterial membrane proteomic workflows.     

Automated integration of monolith-based protein separation with on-plate digestion for mass spectrometric analysis of esophageal adenocarcinoma human epithelial samples

A unique approach of automating the integration of monolithic capillary HPLC-based protein separation and on-plate digestion for subsequent MALDI-MS analysis has been developed. All liquid-handling procedures were performed using a robotic module. This automated high-throughput method minimizes the amount of time and extensive labor required for traditional in-solution digestion followed by exhaustive sample cleanup and analysis. Also, precise positioning of the droplet from the capillary HPLC separation onto the MALDI plate allows for preconcentration effects of analytes for improved sensitivity. Proteins from primary esophageal Barrett's adenocarcinoma tissue were prefractionated by chromatofocusing and analyzed successfully by this automated configuration, obtaining rapid protein identifications through PMF and sequencing analyses with high sequence coverage. Additionally, intact protein molecular weight values were obtained as a means to further confirm protein identification and also to identify potential sequence modifications of proteins. This simple and rapid method is a highly versatile and robust approach for the analysis of complex proteomes.     

Phosphoproteome analysis of mouse liver using immobilized metal affinity purification and linear ion trap mass spectrometry

Since protein phosphorylation is a dominant mechanism of information transfer in cells, there is a great need for methods capable of accurately elucidating sites of phosphorylation. In recent years mass spectrometry has become an increasingly viable alternative to more traditional methods of phosphorylation analysis. The present study used immobilized metal affinity chromatography (IMAC) coupled with a linear ion trap mass spectrometer to analyze phosphorylated proteins in mouse liver. A total of 26 peptide sequences defining 26 sites of phosphorylation were determined. Although this number of identified phosphoproteins is not large, the approach is still of interest because a series of conservative criteria were adopted in data analysis. We note that, although the binding of non-phosphorylated peptides to the IMAC column was apparent, the improvements in high-speed scanning and quality of MS/MS spectra provided by the linear ion trap contributed to the phosphoprotein identification. Further analysis demonstrated that MS/MS/MS analysis was necessary to exclude the false-positive matches resulting from the MS/MS experiments, especially for multiphosphorylated peptides. The use of the linear ion trap considerably enabled exploitation of nanoflow-HPLC/MS/MS, and in addition MS/MS/MS has great potential in phosphoproteome research of relatively complex samples.     

Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.     

Phosphopeptide enrichment: Development of magnetic solid phase extraction method based on polydopamine coating and Ti-IMAC

Protein post translational modifications currently represent one of the main challenges with proteomic analysis, due to the important biological role they play within cells. Protein phosphorylation is one of the most important, with several approaches developed for phosphopeptides enrichment and analysis, essential for comprehensive phosphoproteomic analysis. However, the development of new materials for phosphopeptides enrichment may overcome previous drawbacks and improve enrichment of such peptides. In this regard, new magnetic stationary phases based on polydopamine coating and Ti⁴⁺ immobilization exploit the potential of IMAC enrichment and couple it with the versatility of magnetic solid phase extraction. In this work the use of such stationary phase was extended from the MALDI proof of concept stage with the development of an optimized method for phosphopeptides enrichment compatible with typical shotgun proteomics experimental workflows. Different loading and elution buffers were tested to improve phosphopeptides recovery and enrichment selectivity. Finally, the analysis of isolated peptides pointed out that polydopamine alone is an ideal support matrix for polar post translational modifications because it enables to reduce unspecific binding and preferentially binds hydrophilic peptides.     

Integrating proteomics with electrochemistry for identifying kinase biomarkers

We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers. Our approach combines phosphoproteomics for high throughput cancer-related biomarker discovery from patient tissues and an impedimetric kinase activity biosensor for sensitive validation. Using non-small-cell lung cancer (NSCLC) as a proof-of-concept study, label-free quantitative phosphoproteomic analysis of a pair of cancerous and its adjacent normal tissues revealed 198 phosphoproteins that are over-phosphorylated in NSCLC. Among the differentially regulated phosphorylation sites, the most significant alteration was in residue S165 in the Hepatoma Derived Growth Factor (HDGF) protein. Hence, HDGF was selected as a model system for the electrochemical studies. Further motif-based analysis of this altered phosphorylation site revealed that extracellular-signal-regulated kinase 1/2 (ERK1/2) are most likely to be the corresponding kinases. For validation of the kinase–substrate pair, densely packed peptide monolayers corresponding to the HDGF phosphorylation site were coupled to a gold electrode. Phosphorylation of the monolayer by ERK2 and dephosphorylation by alkaline phosphatase (AP) were detected by electrochemical impedance spectroscopy (EIS) and surface roughness analysis. Compared to other methods for quantification of kinase concentration, this label-free electrochemical assay offers the advantages of ultra-sensitivity as well as higher specificity for the detection of cancer-related kinase–substrate pair. With implementation of multiple kinase–substrate biomarker pairs, we expect this integrated approach to become a high throughput platform for discovery and validation of phosphorylation-mediated biomarkers.     

Phosphoproteome analysis of human liver tissue by long‐gradient nanoflow LC coupled with multiple stage MS analysis

Reversible protein phosphorylation plays a critical role in liver development and function. Comprehensively cataloging the phosphoproteins and their phosphorylation sites in human liver tissue will facilitate the understanding of physiological and pathological mechanisms of liver. Owing to lacking of efficient approach to fractionate phosphopeptides, nanoflow‐RPLC with long‐gradient elution was applied to reduce the complexity of the phosphopeptides in this study. Two approaches were performed to further improve the coverage of phosphoproteome analysis of human liver tissue. In one approach, ten‐replicated long‐gradient LC‐MS/MS runs were performed to analyze the enriched phosphopeptides, which resulted in the localization of 1080 phosphorylation sites from 495 proteins. In another approach, proteins from liver tissue were first fractionated by SDS‐PAGE and then long‐gradient LC‐MS/MS analysis was performed to analyze the phosphopeptides derived from each fraction, which resulted in the localization of 1786 phosphorylation sites from 911 proteins. The two approaches showed the complementation in phosphoproteome analysis of human liver tissue. Combining the results of the two approaches, identification of 2225 nonredundant phosphorylation sites from 1023 proteins was obtained. The confidence of phosphopeptide identifications was strictly controlled with false discovery rate (FDR)≤1% by a MS²/MS³ target‐decoy database search approach. Among the localized 2225 phosphorylated sites, as many as 70.07% (1559 phosphorylated sites) were also reported by others, which confirmed the high confidence of the sites determined in this study. Considering the data acquired from low accuracy mass spectrometer and processed by a conservative MS²/MS³ target‐decoy approach, the number of localized phosphorylation sites obtained for human liver tissue in this study is quite impressive.     

Identification of phosphoproteins and determination of phosphorylation sites by zirconium dioxide enrichment and SELDI-MS/MS

Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.     

Mass spectrometric characterization of stathmin isoforms separated by 2D PAGE

In proteome analysis, the determination of the phosphorylation status of proteins and protein isoforms, which have been separated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), is of prime importance in addition to their identification. In this study, the extent to which such information can be directly extracted from the mass spectrometric data used for identification was evaluated. By searching for metastable peaks which are characteristic for loss of phosphoric acid, the Ser-phosphorylated peptides were identified with a high success rate in reflector matrix-assisted laser desorption/ionization (MALDI) mass maps of in-gel digested proteins. Furthermore, by employing a double enzymatic strategy using trypsin and Glu-C in parallel, improved sequence coverage and additional separation of the potential phosphorylation sites of the isoforms were achieved. The precise location of the modified sites within an identified phosphopeptide was obtained by submitting the corresponding molecular ions directly to nano-electrospray tandem mass spectrometric analysis. In this way the detailed phosphorylation status of six isomers of stathmin separated by 2D PAGE was determined. Two of these six isomers were phosphorylated at all four known sites (serines 15, 24, 37 and 62) and were probably derived from the previously reported alpha and beta forms, which differ by a yet unknown modification. In addition, isomers phosphorylated at serines 15, 24 and 37, serines 24, 37 and 62, serines 24 and 37 and serine 37 only were characterized.     

Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO₂) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/α-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.     

Identification of nuclear proteins of small cell lung cancer cell line H82: An improved procedure for the analysis of silver-stained proteins

An efficient method for digestion and extraction of proteolytic peptides from silver-stained proteins was applied to the characterization of nuclear proteins from the small cell lung cancer H82 (ATCC HTB 175) cell line previously separated by high-resolution large format two-dimensional gel electrophoresis. From 68 spots, evenly distributed on the gel area and representing a wide range of spot intensities, 63 (92%) were successfully identified by matrix-assisted laser desorption/ionization (MALDI) or electrospray ionozation-mass spectrometry (ESI-MS). In five cases where the identification was not possible, the presence of an intense background apparently due to the leakage of polymers from the microtubes or other plastics, was detected. Extensive analysis of peptide sequences by ESI MS/MS experiments allowed the identification of post-translational modifications, such as acetylation, phosphorylation, deamidation of asparagine residues and the presence of isoaspartic acid. A new protein variant not reported in sequence databases was also detected.     

Mass spectrometric analysis of protein phosphorylation

Phosphorylation is one of the most common posttranslational modifications of proteins in eukaryotic cells; it plays an important role in a wide spectrum of biological processes. This makes its study an important task for understanding cell functioning mechanisms. The aim of phosphoproteomics is a global mass spectral analysis of the phosphoprotein composition of cells, i.e., phosphoproteome. Nowadays, new effective methods are actively developed, which succeed not only in the detection of phosphorylated proteins but also in the determination of phosphorylated amino acid residues (phosphorylation sites) and in the quantitative comparison of phosphorylation among several specimens. Despite the analysis of protein phosphorylation remains a complicated problem, the available methods nowadays allow the detection of thousands of phosphorylation sites in the very same experiment. The present review covers the main methods utilized in contemporary phosphoproteomics: phosphoprotein and phosphopeptides enrichment as well as the mass spectrometric analysis of protein phosphorylation.