Application of reversed phase high performance liquid chromatography for subproteomic analysis of cardiac muscle

Abstract

The application of protein separation methodologies, such as reversed phase chromatography, should allow differential separation of the proteome, or at least specific subproteomes, comparable to that achieved by two-dimensional electrophoresis (2-DE). A rapid sequential protein extraction method (termed "IN Sequence") was developed to isolate three distinct subproteomes of cardiac muscle. Two subproteomes, those enriched for the cytoplasmic or myofilament proteins, can be separated by either reversed phase high performance liquid chromatography (RP-HPLC) or 2-DE. Reversed phase HPLC of the myofilament protein enriched extract was optimized for resolution and peak numbers by altering flow rate, gradient rate and the organic modifiers, isopropanol and acetronitrile. The myofilament protein enriched extract from failing swine heart, due to coronary artery ligation (LAD), was compared to the extract from a sham operated animal (SHAM). The HPLC chromatograms of these extracts were similar, but distinctive in many regions. The HPLC fractions, collected within some of these distinct regions of the chromatograms were analyzed using peptide mass fingerprinting - mass spectrometry and immunoblot analysis. Two myofilament proteins, troponin T and myosin heavy chain, were identified and found differentially modified in the SHAM and LAD hearts. Both troponin T and myosin heavy chain are problematic proteins for 2-DE, but yet they were resolved by reversed phase chromatography. Therefore, RP-HPLC can be used in conjunction with 2-DE to enhance protein separation of myofilament protein subproteome.

Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics

“…The myofilament extraction method employed in this study was based on a previous method developed by Van Eyk and co-workers (Arrell et al 2001; Neverova and Van Eyk 2002). It consists of two extraction steps: first extraction using HEPES buffer at neutral pH to extract cytosolic proteins and the second extraction using 1% TFA (pH 2.2) to extract myofilament proteins from the remaining pellet of the first extraction.…”

A Novel, In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain

“…Nevertheless, there have been a few cases where RP-HPLC has been utilized at the protein level as the second dimension in tandem for two-dimensional protein separation methods following ion exchange chromatography, chromatofocusing, electroelution, or size exclusion chromatography (20 -24), although these tandem technologies have not been routinely applied to cardiac muscle proteins. However, separate from two-dimensional LC applications, RP-HPLC has been used alone to separate sarcomeric proteins from failing versus non-failing swine hearts by Neverova and Van Eyk (25). Following buffer optimization, they were able to enhance separation of myofilament proteins and identify alterations in MHC in heart failure.…”

Role of chromatographic techniques in proteomic analysis

“…The most common approach today is to divide the cell into parts or subproteomes, thereby simplifying protein separation and identification [71]. Even so, subproteomes such as mitochondria, nucleus, cytoplasmic/soluble cellular pools can have 1000's of proteins.…”