Mapping intact protein isoforms in discovery mode using top-down proteomics.

A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This 'bottom-up' process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species. 'Top-down' interrogation of whole proteins can overcome these problems for individual proteins, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research.

High-mobility group A1 (HMGA1) protein expression correlates with cisplatin-induced cell death in squamous cell carcinoma of skin.

It is well known that HMGA1 group of non-histone chromosomal proteins are up-regulated in several human cancers. We studied the HMGA1 expression in squamous cell carcinoma of skin in mice followed by the treatment with Cisplatin, which is often used in combination therapies of cancers. A short course of Cisplatin treatment led to apoptotic cell death and downregulation (by 40%) of HMGA1. However, extended treatment of Cisplatin caused necrotic cell death; concomitantly HMGA1 expression decreased by 90%. Present results indicate a strong association of HMGA1 with Cisplatin-linked tumor regression. Therefore, HMGA1 could be a potential target in designing therapeutic strategies against cancer.

Mass spectrometric analysis of high-mobility group proteins and their post-translational modifications in normal and cancerous human breast tissues.

High-mobility group (HMG) A1 proteins including HMGA1a and HMGA1b are chromosomal proteins that function in a variety of cellular processes such as cell growth, transcription regulation, neoplastic transformation, and progression. Overexpression of HMGA1 proteins has been associated with almost every type of cancer cells. Post-translational modifications (PTMs) of HMGA1 proteins in different types of human cancer cell lines have been extensively explored over the past decade. Here, we extended the identification of PTMs of HMGA1 proteins to human breast tumor tissue specimens with different carcinoma progression stages (metastatic and primary cancer) as well as the paired adjacent normal breast tissues. In this regard, we employed tandem mass spectrometry to examine the nature and sites of PTMs of HMGA1 proteins isolated from cancerous/normal human breast tissues. Novel PTMs of HMGA1a protein, that is, monomethylation at Lys30 and Lys54 as well as monophosphorylation at Ser43 and Ser48, were detected in cancer tissues. In these cancer tissues, we also found C-terminal constitutive phosphorylation in HMGA1a and HMGA1b as well as mono- and dimethylation of Arg25 in HMGA1a, which were previously found to be present in these proteins isolated from human cancer cell lines. Furthermore, a more complex spectrum of PTMs on HMGA1 proteins was correlated with a more aggressive malignancy in human breast cancer tissues.

Increased expression of high mobility group A proteins in lung cancer.

High mobility group A (HMGA) proteins play an important role in the regulation of transcription, differentiation, and neoplastic transformation. In this work, the expression of HMGA 1 and 2 in 152 lung carcinomas of mainly non-small-cell histological type has been studied by immunohistochemistry in order to evaluate their feasibility as lung cancer markers. In 17 lung cancer cases, the related bronchial epithelial changes were also studied for HMGA1 and 2 expression. RNA expression of HMGA1a and b isoforms and of HMGA2 was determined by real-time semi-quantitative RT-PCR in 23 lung carcinomas. High expression of HMGA1 and HMGA2 at both mRNA and protein levels was detected in lung carcinomas, compared with normal lung tissue. Nuclear immunostaining for HMGA1 and 2 proteins also occurred in hyperplastic, metaplastic, and dysplastic bronchial epithelium. Increased nuclear expression of HMGA1 and 2 correlated with poor survival (for adenocarcinomas, HMGA1, p=0.006; HMGA2, p=0.05). While the expression of HMGA2 was significantly associated with cell proliferation (p=0.008), HMGA1 expression did not show any association with proliferation or apoptotic index. Sequencing of HMGA2 transcripts from tumours with very high expression showed a normal full-length transcript. As HMGA proteins were expressed in about 90% of lung carcinomas and their expression was inversely associated with survival, they may provide useful markers for lung cancer diagnosis and prognosis.