Here, we present a two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) to separate and identify human pituitary adenoma tissue proteome, which presents a good and reproducible 2DE pattern. Many proteins are observed in each 2DE spot when analyzing complex cancer proteome with the use of high-sensitivity MS.
Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may be classified as either clinically functional or nonfunctional PA (FPA and NFPA). NFPA is difficult for early stage diagnosis and therapy due to barely elevating hormones in the blood compared to FPA. Our long-term goal is to use proteomics methods to discover reliable biomarkers for clarification of PA molecular mechanisms and recognition of effective diagnostic, prognostic markers and therapeutic targets. Effective two-dimensional gel electrophoresis (2DE) coupled with mass spectrometry (MS) methods were presented here to analyze human PA proteomes, including preparation of samples, 2D gel electrophoresis, protein visualization, image analysis, in-gel trypsin digestion, peptide mass fingerprint (PMF), and tandem mass spectrometry (MS/MS). 2-Dimensional gel electrophoresis matrix-assisted laser desorption/ionization mass spectrometry PMF (2DE-MALDI MS PMF), 2DE-MALDI MS/MS, and 2DE-liquid chromatography (LC) MS/MS procedures have been successfully applied in an analysis of NFPA proteome. With the use of a high-sensitivity mass spectrometer, many proteins were identified with the 2DE-LC-MS/MS method in each 2D gel spot in an analysis of complex PA tissue to maximize the coverage of human PA proteome.
PA is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, which play important roles in the human endocrine system. PA includes clinically functional and nonfunctional PAs (FPA and NFPA)1,2. NFPA is difficult in early stage diagnosis and therapy because of only slightly elevated hormone levels (e.g., LH, and FSH) in blood compared to FPA, which has significantly increased levels of corresponding hormones in blood3,4,5. The clarification of molecular mechanisms and discovery of effective biomarkers has important clinical significance in the diagnosis, therapy, and prognosis of NFPA. Our long-term goal is to develop and use proteomic methods to study NFPA for the discovery of reliable biomarkers to clarify its molecular mechanisms, and recognize effective therapeutic targets as well as diagnostic and prognostic markers. 2-DE coupled with MS methods have been extensively used in our long-term research program regarding human PA proteome1,2,6,7, including establishment of proteome reference maps3,8, analysis of differentially expressed protein profiles9,10,11,12,13, hormone variants14,15, post-translational modifications such as phosphorylation14 and tyrosine nitration16,17,18, the proteomic variation of invasive relative to noninvasive NFPAs19, and the proteomic heterogeneity of NFPA subtypes13, which led to the discovery of multiple important pathway networks (mitochondrial dysfunction, cell cycle dysregulation, oxidative stress, and MAPK signaling system abnormality) that are altered in NFPA13,19,20.
2DE separates proteins according to their isoelectric point (pI) (isoelectric focusing, IEF) and molecular weight (via sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE)1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23. This is a common and classical separation technique in the field of proteomics, since the introduction of the concepts of the proteome and proteomics in 199524. MS is the crucial technique for finding out the identity of 2DE-separated proteins, including PMF and MS/MS strategies. The very rapid development of MS instruments, especially in the aspects of detection sensitivity and resolution, in combination with the improvement of LC system, greatly improves the identity of low or extremely low abundance proteins in a proteome to maximize the coverage of a proteome. It also challenges our traditional concepts that only one or two proteins are present in a 2D gel spot in an analysis of complex human tissue proteome and provides an opportunity to identify multiple proteins in a 2D gel spot in an analysis of complex human tissue proteome and maximize the coverage of NFPA proteome.
Here we describe detailed protocols of 2DE-MALDI MS PMF, 2DE-MALDI MS/MS, and 2DE-LC-MS/MS which have been successfully used in the analysis of human NFPA proteome. The protocols include preparation of samples, first dimension (isoelectric focusing, IEF), second dimension (SDS-PAGE), visualization of proteins (silver staining and Coomassie blue staining), image analysis of 2D gel, in-gel trypsin digestion, purification of tryptic peptides, PMF, MS/MS, and database searing3,8,25,26. Moreover, this protocol easily translates for the analysis of other human tissue proteomes.
The present protocol follows the guidelines of the Xiangya Hospital Medical Ethics Committee of Central South University, China. A head cap and gloves should be worn for the entire experimental procedure to avoid keratin contamination from skin and hair8.
1. Preparation of Samples
2. Two-Dimensional Gel Electrophoresis
3. Identification of 2DE-Separated Proteins with MS
1. 2DE-MALDI MS PMF: With the experimental procedure described above, a total of 150 µg proteins were extracted from FSH-expressed NFPA tissues (female; 50 years old, ACTH (-), GH (-), PRL (-), LH (-), FSH (+), and TSH (-)) and arrayed with an 18 cm IPG strip (pH 3-10 NL) and a large-format SDS-PAGE gel, then visualized with silver staining. We obtained a reproducible and good 2DE gel pattern of NFPA tissue proteome (Figure 1), with an average positional deviation of 1.98 ± 0.75 mm in the IEF direction and 1.62 ± 0.68 mm in the SDS-PAGE direction, and with an approximately 1,200 protein spots detected in the 2DE gel map that were mainly distributed within the area of pH 4-8 and mass 15-150 kDa3. In total, 337 gel spots were excised and subjected to gel destaining, trypsin digestion, purification of tryptic peptides, and MALDI-TOF-MS PMF analysis. PMF data was used for protein identity with MASCOT search against the human protein database. A total of 192 redundant proteins (representing 107 nonredundant proteins) were identified from 141 gel spots out of 337 analyzed gel spots (141/337 = 42%), and no protein was identified in 196 spots (196/337 = 58%) (Table 1). For those 141 spots, only one protein per spot was identified in 121 spots, 2 proteins per spot were identified in 12 spots (Spots 31, 32, 33, 41, 42, 43, 44, 68, 75, 137, 148, and 224), 5 proteins per spot were identified in 3 spots (Spots 19, 22, and 23), 6 proteins per spot were identified in 3 spots (Spots 24, 91, and 93), and 7 proteins per spot were identified in 2 spots (Spots 72, and 92).
2. 2DE-MALDI MS/MS and 2DE-LC-MS/MS: Using the experimental procedure described above, a total of 500 µg of protein extracted from an invasive NFPA tissue (male, 22 years old, ACTH (-), hGH (-), PRL (++), LH (-), and FSH (-)) was arrayed with an 18 cm IPG strip (pH 3-10 NL) and a large-format SDS-PAGE gel, then visualized with Coomassie Blue G250 staining. This staining yielded a reproducible and high quality 2DE gel pattern of invasive NFPA tissue proteome (Figure 2), with an average positional deviation of 1.95 ± 0.85 mm in the IEF direction and 1.85 ± 0.79 mm in the SDS-PAGE direction, and with approximately 1,100 protein spotsdetected in the 2DE gel map that were mainly distributed within the range of pH 4-8 and mass 15-150 kDa29. The 10 spots randomly selected and labeled in Figure 2 were excised and subjected to gel destaining, trypsin digestion, purification of tryptic peptides, followed by MALDI-MS/MS and LC-ESI-MS/MS analyses. Each MS/MS dataset was used for protein identity with MASCOT search against the human protein database. For all spots, an average of 1 protein per spot was identified with MALDI-TOF-TOF MS/MS analysis, whereas many proteins (an average of 63 proteins in a single gel spot, an average of 71 proteins in a pool of 2 matched spots, and an average of 118 proteins in a pool of 3 matched gel spots) in each corresponding spot were identified with LC-ESI-MS/MS analysis (Table 2). Here, one must mention that the LC-ESI-MS/MS system has a much higher sensitivity and resolution relative to the MALDI-MS/MS analysis system. This data clearly demonstrates that each 2D gel spot contains many proteins, not just one or two proteins in the complex human cancer tissue proteome.
Figure 1: Silver stained 2-DE pattern of an FSH-expressed NFPA proteome analyzed with IPGstrip pH 3–10 NL and 12% gel concentration of SDS-PAGE. The red-brown spots are the silver-stained proteins, the orange is the background of silver-stained gel. The labeled spots were analyzed with MALDI-TOF-MS PMF. Reproduced from Wang X, et al. (2015)3, with permission from Wiley-VCH. Please click here to view a larger version of this figure.
Figure 2: Coomassie blue stained 2-DE pattern of an NFPA proteome analyzed with IPGstrip pH 3-10 NL and 12% gel concentration of SDS-PAGE. The labeled spots were analyzed with MALDI-TOF-TOF MS/MS and LC-ESI-MS/MS. Spots 1*, 2*, 3*, 5*, and 16* were matched to the corresponding spots 1, 2, 3, 5, and 16, and pooled from 2 matched gels. Modified from Zhan X, et al. (2018)29, with permission from Wiley-VCH. Please click here to view a larger version of this figure.
Table 1: The number of proteins that were identified in each 2DE gel spot with MALDI-TOF-MS PMF analysis.
Table 2: The number of proteins that were identified in each 2DE gel spot with MS/MS analysis.
2DE, coupled with MS methods including 2DE-MS PMF and 2DE-MS/MS, has been successfully used in our long-term program – the use of proteomics to study human NFPA proteomic variations and molecular network variations for the elucidation of molecular mechanisms and discovery of effective biomarkers for NFPAs. 2DE-based comparative proteomics with good reproducibility plays an important role in the identification of NFPA proteomic variations9,10,11,12,13,19, and 2DE coupled with the antibody method demonstrates its advantages in the detection of a given post-translational modification and variants of a given protein, such as detection of tyrosine nitration16,17,18 and GH variants14,15.
However, 2DE coupled with a MS method is considered a labor-intensive technique with difficulties in analyses of low abundance proteins, hydrophobic proteins, extremely basic or acidic proteins, and extremely low or high mass proteins2. For instance,extremely low-mass (<10 kDa) or high-mass (>150 kDa) proteins, and extremely basic (pI>7.5 or 8) or acidic (pI<3.5 or 4) proteins are not easily detected by 2DE with an 18-cm IPG strip of pH 3-10 NL3. With the rapid development of gel-free separation methods, including two-dimensional liquid chromatography (2D-LC) coupled with stable isotope labeling such as iTRAQ and TMT in the last ten years23, it seems that 2-DE has gradually withdrawn from its central status in proteomics research in the analysis of the complex tissue proteome because 2D-LC coupled with stable isotope labeling and MS/MS methods effectively overcome the drawbacks of 2DE-based methods.
Recent studies found that, with the use of high-sensitivity mass spectrometry, such as an LC-ESI-MS/MS (its sensitivity is in 1-10 amol range) in the identification of 2DE-separated proteins, the new characteristics of the 2-DE method were discovered which show that a 2D gel spot contains many proteins (Table 2). This ran counter to the traditional concept of only one or two proteins per spot, as is evidenced by the World 2DPAGE database (World-2DPAGE portal: http://world-2dpage.expasy.org/portal). This clearly demonstrates that the throughput of 2-DE in the identity of proteins in a complex human proteome is much higher than one previously assumed, namely that there were only one to two proteins in a 2D gel spot. This techinique offers the promise of use for 2DE in the field of proteomics. One 2DE can separate over a thousand to ten thousand spots3,8,22 in the analysis of the complex human proteome, thus 2DE is a more detailed separation technique to maximally simplify the complexity of proteome samples before LC-MS/MS analysis relative to the 2D-LC system that the first-dimensional LC commonly generates 18 to 30 fractions before LC-MS/MS analysis. Therefore, 2DE in combination with high-sensitivity mass spectrometry can be used to establish the tremendous information reference map for the complex human proteome, especially for those low or extremely low abundance proteins. In addition, 2-DE in combination with stable isotope labeling (such as iTRAQ, TMT, and SILAC) and high-sensitivity mass spectrometry can be used to quantify large-scale proteomic variations among different conditions. Moreover, 2DE coupled with stable isotope labeling and high-sensitivity mass spectrometry has absolute advantages in large-scale detection and quantification of protein PTMs, protein variants, protein speciation, and protein species. These advantages will have 2DE revitalized in the field of proteomics.
Furthermore, the present protocols for the analysis of human PA tissue proteome described here are easily translated to study other human disease proteomes.
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (Grant No. 81572278 and 81272798 to XZ), the grants from China "863" Plan Project (Grant No. 2014AA020610-1 to XZ), the Xiangya Hospital Funds for Talent Introduction (to XZ), and the Hunan Provincial Natural Science Foundation of China (Grant No. 14JJ7008 to XZ). The authors also acknowledge the scientific contribution of Dr. Dominic M. Desiderio in the University of Tennessee Health Science Center. X.Z. continuously developed and used 2DE-MS methods to analyze pituitary adenoma proteome starting from 2001, conceived the concept for the present manuscript, wrote and revised the manuscript, coordinated the pertinent work, and was responsible for the financial support and corresponding work. Y.L. participated in revision of the manuscript. Y.H participated in collection of references, and revision of the manuscript. All authors approved the final manuscript.
Ettan IPGphor 3 | GE Healthcare | isoelectric focusing system. | |
Ettan DALTsix multigel caster | Amersham Pharmacia Biotech, Piscataway, NJ, USA | ||
Ettan DALT II System | Amersham Pharmacia Biotech, Piscataway, NJ, USA | The vertical electrophoresis system | |
Ettan IPGphor strip holder | Amersham Pharmacia Biotech, Piscataway, NJ, USA | ||
Ettan DALTsix multigel caster | Amersham Pharmacia Biotech, Piscataway, NJ, USA | Multigel caster | |
Voyager DE STR MALDI-TOF MS | ABI, Foster City,CA | MALDI-MS PMF | |
MALDI-TOF-TOF | Autoflex III, Bruker | MALDI-MS/MS mass spectrometer | |
LTQ-OrbiTrap Velos Pro ETD | Thermo Scientific, Waltham, MA, USA | ESI-MS/MS mass spectrometer | |
EASY-nano LC system | Proxeon Biosystems, Odense, Denmark | High performance liquid chromatography system | |
PepMap C18 trap column | 300 μm i.d. × 5 mm length; Dionex Corp., Sunnyvale, CA, USA | ||
RP C18 column | 75 μm i.d., 15 cm length; Dionex Corp., Sunnyvale, CA, USA | ||
KimWipe | Kimvipe | Insoluble paper towel | |
Watter | Made by PURELAB flex instrument | ||
Polytron Model P710/35 homogenizer | Brinkmann Instruments, Westbury, NY | ||
PDQuest | Bio-Rad, Hercules, CA | 2D gel image analysis software | |
SEQUEST | Thermo Proteome Discoverer 1.3 (version No. 1.3.0.339) | ||
DataExplore (ver. 4.0.0.0) software | MS spectrum-processing software | ||
Mascot software | PMF-based protein searching software | ||
Mascot software | MS/MS-based protein searching software | ||
Proteome Discoverer software v.1.3 beta | Thermo Scientific | ||
Xcalibur software v.2.1 | MS/MS data-acquired management software | ||
Uniprot version 201410.1_HUMAN.fasta | Human protein database | ||
SEQUEST (version No. 1.3.0.339) | MS/MS-based protein searching software I | ||
MASCOT (version 2.3.02) | MS/MS-based protein searching software II | ||
C18 ZipTip microcolumn | Millipore | ||
PeptideMass Standard kit | Perspective Biosystems | ||
Pierce BCA Protein Assay Kit | Thermo Fisher Scientific | 23227 | |
2-D Quant Kit | GE Healthcare | 80-6483-56 | |
BIS-ACRYLAMIDE | AMRESCO | 0172 | |
ACRYLAMIDE | AMRESCO | 0341 | |
DTT | Sigma-Aldrich | D0632 | |
Thiourea | Sigma-Aldrich | T8656 | |
Urea | VETEC | V900119 | |
SDS | AMRESCO | 0227 | |
CHAPS | AMRESCO | 0465 | |
TEMED | AMRESCO | M146 | |
Ammonium Persulfate | AMRESCO | M133 | |
Trypsin | Promega, Madison, WI, USA | V5111 | |
IPG buffer pH 3-10, NL | GE Healthcare | 17-6000-87 | |
Immobiline Dry Strip pH 3-10NL,18cm | GE Healthcare | 17-1235-01 |