Reliable FASP-based procedures for optimal quantitative proteomic and phosphoproteomic analysis on samples from acute myeloid leukemia patients

Abstract

BackgroundSatisfactory sample preparation for mass spectrometry-based analysis is a critical step in the proteomics workflow. The quality and reproducibility of sample preparation can determine the coverage and confidence of proteomics results. Up to date, several methodologies have been described to produce suitable peptides for mass spectrometry analysis, followed by strategies for enrichment of post-translational modified peptides, if desired. Among them, the filter-aided sample preparation (FASP) has been introduced as a method to allow for removal of denaturants, reductants, alkylators, lipids and nucleic acids prior to trypsin digestion. Despite the high proteolytic digestion and contaminant removal efficiency described for this method, filter failure and consequently complete sample loss can discourage the use of this approach by the proteomic community.ResultsAs judged by our quality controls, we were able to perform reliable and reproducible FASP for mass spectrometry analysis that allowed the quantification of 2141 proteins and 3694 phosphopeptides from as little as 20 and 320 μg of protein lysate from acute myeloid leukemia (AML) patients, respectively. Using the immobilized metal ion affinity chromatography (IMAC) method resulted in samples specifically enriched in phosphopeptides and allowed the quantification of a high number of both di- and multi-phosphopeptides in addition to the abundant mono-phosphopeptides. The workflows’ high reproducibility from three biological replicates was demonstrated by the similar number of quantified proteins and localized phosphosites, and confirmed by the similar distributions of their molecular functions. We found that the combination of the FASP procedure with StageTip mixed-mode fractionation and IMAC are excellent workflows for the reproducible and deep study of AML proteomes and phosphoproteomes, respectively.ConclusionsThe FASP procedure can be carried out without the risk of filter failure by performing a simple test of the filter quality before adding the protein sample. Herein, we demonstrate an efficient and reproducible FASP-based pipeline for the proteomic and phosphoproteomic analysis of AML patient samples which also can be used for the analysis of any other protein samples.Electronic supplementary materialThe online version of this article (doi:10.1186/s12575-016-0043-0) contains supplementary material, which is available to authorized users.

Proteome and phosphoproteome changes associated with prognosis in acute myeloid leukemia

“…Sample preparation of patient cell lysate in 4% sodium dodecyl sulfate (SDS)/0.1 M Tris-HCl (pH 7.6) and immobilized metal affinity chromatography (IMAC) has been described earlier. 31 Briefly, 20 µg of each patient lysate was prepared both as 1) a label-free sample and 2) mixed with 10 µg of the super-SILAC mix for proteomic analyses, and processed according to the filteraided sample preparation (FASP) protocol 31,32 (supplemental Methods; Figure 1C). The super-SILAC spiked peptide samples were fractionated using styrenedivinylbenzene-reversed phase sulfonate (SDB-RPS) plugs (Empore, 3M).…”

Selecting Sample Preparation Workflows for Mass Spectrometry-Based Proteomic and Phosphoproteomic Analysis of Patient Samples with Acute Myeloid Leukemia

“…The multi-steps of the FASP procedure together with increasing reports of filter failure [25,32] have discouraged the use of this strategy in the proteomics community despite its proven efficiency as in our comparison study. The use of faulty filters in the FASP protocol will cause the loss of most of the proteins in the sample as they are not retained on the filter membrane, yielding a poor peptide recovery after proteolytic digestion [32].…”

“…The use of faulty filters in the FASP protocol will cause the loss of most of the proteins in the sample as they are not retained on the filter membrane, yielding a poor peptide recovery after proteolytic digestion [32]. A reduced centrifugal speed has been suggested to avoid FASP failure [25].…”

“…A reduced centrifugal speed has been suggested to avoid FASP failure [25]. However, an initial simple quality filter test with the urea FASP buffer worked best in our hands to differentiate working from failing protein filters [32]. …”

Quick 96FASP for high throughput quantitative proteome analysis

“…Following protein digestion, FASP offers the additional advantage of further peptide cleanup due to the fact that the latter pass the membrane while other macromolecules are retained on the filter membrane in the final centrifugation step. FASP has thus been widely applied to various types of samples including body fluids and lysates of animal or plant tissues, fungi and bacteria [10-15]. Further developments of FASP methods have also been reported, such as MED-FASP and eFASP to improve peptide recovery and efficiency [16, 17], iFSP to accommodate chemical labeling reactions [18], microwave-assisted FASP to speed up on-filter digestion [19], abFASP to analyze affinity-purified protein complexes [20], endoProteoFASP to study the peptidome [21], and other adaptations to investigate specific subsets of modified proteins/peptides [22, 23].…”